Synergistic stimulation of mucociliary clearance to treat mucus obstruction in cystic fibrosis and other mucus-obstructive disorders

Abstract

The present disclosure provides a method of treating an individual suffering from a mucosal obstructive disease, the method comprising administering to the individual a β-adrenergic agonist or an adenylate cyclase activator in combination with a cholinergic agonist to treat the mucosal obstructive disease.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 408,596, filed September 21, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Cystic fibrosis (CF) is a multisystem syndrome whose most significant clinical phenotypes are airway mucus obstruction, chronic lung infection, and neutrophilic inflammation. Without treatment, the resulting tissue damage leads to a lifelong decline in lung function. CF is caused by loss-of-function mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel critical for airway fluid secretion. While CF airways appear normal at the time of production, the ability of airway surface liquid (ASL) to kill bacteria is impaired (1), and mucus clearance is slowed (2). Chronic lung infection is a major cause of lung function decline in humans (3, 4). However, transgenic CF ferrets ("CF ferrets") persist with a mucosal obstructive phenotype, characterized by bronchial obstruction and inflammation, despite antibiotic prophylaxis against chronic infection (5). Summary of the Invention [Problem to be solved by the invention]

[0003] Improving airway mucociliary clearance (MCC) is an important therapeutic goal in CF. Improvements in mucus clearance have been achieved through inhaled therapy with recombinant human DNase (Pulmozyme) (6, 7), hypertonic saline (8, 9), or powdered mannitol (10), and in some cases are sufficient to demonstrate clinical benefit. For most CF patients, the most effective improvement in mucus clearance is achieved through small-molecule modulators that partially restore function in CFTR with specific mutations (11, 12). For patients with mutations that cannot be treated with current modulators or who experience reduced lung function despite modulators, further improvement in mucus clearance may be therapeutic. MCC is a function of the volume and composition of airway surface liquid (ASL), the rheological properties of secreted mucus, and cilia beat frequency (CBF) (13). [Means for solving the problem]

[0004] Provided herein are methods and compositions for the treatment of cystic fibrosis in an individual in need thereof.

[0005] The present invention provides a method of treating an individual suffering from a mucus obstructive disorder, the method comprising administering to the individual a β-adrenergic agonist or an adenylate cyclase activator in combination with a cholinergic agonist to treat the individual for the mucus obstructive disorder.

[0006] Also provided is a method for increasing the rate of airway submucosal gland secretion in an individual, the method comprising administering to the individual a β-adrenergic agonist or an adenylate cyclase activator in combination with a cholinergic agonist to increase the rate of submucosal gland secretion in the individual.

[0007] The present invention also provides a method for inhibiting cholinergic agonist-induced airway smooth muscle contraction in an individual, the method comprising administering to the individual a β-adrenergic agonist or an adenylate cyclase activator, wherein the administration of the β-adrenergic agonist of the adenylate cyclase activator occurs before or simultaneously with the administration of the cholinergic agonist to inhibit airway smooth muscle contraction.

[0008] Compositions and devices for practicing the subject methods are also provided.

[0009] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Rather, the dimensions of the various features have been arbitrarily increased or reduced for the sake of clarity. The drawings include the following figures: [Brief explanation of the drawings]

[0010] [Figure 1A-B] Synergistic mucus clearance in the trachea of ​​CF ferrets and WT pigs. (A) Time course of MCC rate (MCCV, a measure of the rate of MCC) in CF ferrets in response to the direct adenylate cyclase activator 10 μM forskolin (blue open circles, Fsk, n = 4), the cholinergic agonist 0.3 μM carbachol (red open squares, Carb, n = 4), or their combination (filled symbols). CF ferrets were of five CFTRG551D, one CFTRΔF / ΔF, and one CFTRG551D / KO genotype. (B) Data summarized as boxplots. Bs: basal / unstimulated MCCV; Sum: arithmetic sum of MCCV measurements for agonists used separately; SR: synergistic response, the MCCV measured with the combined agonists. The SR was 5.7-fold greater than the sum, indicating synergy (P = 0.006, n = 3–7). [Figure 1C-D] (C) Same protocol and symbols as for MCCV:CF ferrets in 2- to 5-day-old piglets. (D) Summary data. SR was 3.9-fold greater than the total (P = 3.8E-05, n = 4-8). [Figure 1E-F](E) Responses in pigs using the β-adrenergic agonist 10 μM formoterol / Fmt (blue circle) instead of forskolin, otherwise the same protocol and symbols. (F) Summary data. SR was 3.4-fold greater than the sum (P = 0.005, n = 3–7). [Figure 2A-B] Increased cAMP levels due to adenylate cyclase activation suppressed muscle tone and airway constriction in response to carbachol. (A, B) To measure muscle tension, one end of an isolated WT ferret tracheal muscle bundle was fixed to a Silgard-lined Petri dish filled with KRB solution, and the other end was connected to a pre-calibrated strain gauge with 26-gauge wire. Tension responses to increasing doses of carbachol in the absence (A) and presence (B) of 10 μM forskolin are shown. [Figure 2C-F] (C-F) To measure airway narrowing, thin sections (approximately 2 mm) of tracheal rings from WT piglets and WT or CF ferrets were treated with carbachol alone or in the presence of forskolin (F or Fsk) or formoterol (Fmt). The lumen was imaged over time, and the area was measured as an assay of muscle contraction. After a baseline period (black squares), carbachol (0.3 µM, red squares) was added or in the presence of 10 µM forskolin or formoterol (red closed circles). The average response over 10 min intervals is shown below. (C) Trachea from WT piglets treated with forskolin (n = 3-7). (D) Trachea from WT piglets treated with formoterol (n = 5). (E) Trachea from WT ferrets (2 tracheas, 5 experiments). (F) CF ferrets (n = 2, one CFTRΔF / ΔF and one CFTRΔF / G551D). [Figure 3A-C]Combined agonists synergistically increase gland mucus secretion. Using the same labels as in Figure 1, the average secretion rates for 15 WT pigs (A), 12 WT ferrets (D), and 2 CF ferrets (G) are summarized as boxplots. Each agonist increased secretion above baseline values, and the ratio (SR, synergistic response) to the combined agonist was significantly greater than the arithmetic sum (sum) of the individual responses. (B) Average secretion rates for individual WT pigs in response to 10 μM forskolin alone and in combination with 0.3 μM carbachol (60 glands, 8 pigs). (C) Same as in B, but with carbachol alone and forskolin combined (60–61 glands, 7 pigs). [Figure 3D-F] (E, F) Data from WT adult ferrets under the same conditions as pigs (26–29 glands, 7 ferrets). [Figure 3G-I] (H, I) CF ferret data generated under the same conditions as for pigs and WT ferrets. One ferret was run for each condition, and secretion rates were measured in 7–14 glands. The time course of the average response is plotted at 10-minute intervals. [Figure 4A-B] The combined agonist inhibits sodium absorption by surface epithelium and stimulates anion secretion. (A) Diagram of two electrogenic ion transport pathways across the apical airway epithelium: anion secretion increases surface fluid, while Na+ absorption decreases surface fluid. The two pathways have opposing effects on fluid depth but additive effects on short-circuit current (Isc) due to their opposite valence and transport direction. (B) Raw trace of Isc in pig tracheal epithelium using Chart4 software. After reaching a stable unstimulated Isc (here, more than 2 h after mounting), 10 μM forskolin, 0.3 μM carbachol, 10 μM benzamil, 20 μM benzopyrimidopyrrolooxazinedione (BPO-27), and 200 μM niflumic acid were added sequentially at the indicated times. [Figure 4C-D] (C, D) Pig tracheal mucosa: Mean ΔIsc plots over time for (C) forskolin alone followed by forskolin + carbachol, and (D) reverse order of agonist addition. [Figure 4E-F](E and F) Ferret tracheal mucosa: Mean ΔIsc plots using the same protocol as for pigs. Pig traces (C, D) are based on 10–12 experiments using tissue from six to seven pigs. Ferret traces (E, F) are based on seven experiments using tissue from five ferrets. [Figure 5] Agonists stimulated ciliary beating frequency with additive effects. Ciliary beating frequency was measured in human nasal mucosa from four subjects. Unstimulated tissue CBF (Hz) was 10.46 ± 0.95 in Krebs buffer (KRB) at 37°C. Each agonist alone slightly increased CBF, but the increase was not significant in this small sample (carbachol: 11.04 ± 1.3 (5.3%), forskolin: 12.06 ± 1.22 (9.8%)). When comparing the delta CBF to unstimulated CBF (KRB), the combined agonist significantly increased CBF by 13.31 ± 0.77 (27.2%, n = 4, P < 0.05), but not when comparing the delta CBF to the arithmetic sum of the combined agonist delta CBF: 2.85 ± 0.76 (synergy paradigm) vs. 2.19 ± 0.66 (arithmetic sum) (n = 4, P = 0.47). (Note that the ciliated tissue was studied in Krebs solution in this experiment, which diluted the effects of the agonists on the ASL.) [Figure 6A-B]Schematic diagram correlating increased ASL production with increased mucus clearance. (A) Relationship between ΔIsc, ASL depth, and MCCV. MCCV (redrawn from Figure 1C) is shown in the main graph, and the inset shows ΔIsc (from Figure 4C), with time points aligned to the MCCV graph. The brown dashed line is the estimated change in ASL depth in the absence of MCCV. (B) Diagram of the major electrogenic ion flows across the tracheal surface epithelium in four conditions: baseline, β-adrenergic (β-Adn), carbachol (CCh), and β-Adn + CCh. Each panel displays the estimated state of anion secretion, Na+ absorption, and the resulting change in ASL depth. The change in ASL depth is estimated. In our experiments, the primary change was an increase in the rate of mucus clearance, which tends to counteract the increase in ASL depth. Because carbachol inhibits Na+ (and fluid) absorption and stimulates anion (and fluid) secretion, the combined agonists have opposite effects on Isc but at least an additive increase in ASL depth. [Figure 6C] (C) Schematic diagram of the component processes leading to a synergistic increase in MCCV in the ex vivo trachea of ​​WT ferrets, WT pigs, and CF ferrets. The end result is a significant increase in MCCV. [Figure 7A-D] Individual MCCV responses to the synergy paradigm in seven CF ferrets. (A-G) Protocol and genotype are indicated on each time-MCC rate plot. Note that a reduced y-axis scale is used to display synergistic MCCV (E) and (G). Also note that the mean MCCV for T10-30 to 0.3 µM carbachol in (E) is less than 5% of that in (A) and (C). [Figure 7E-G] Individual MCCV responses to the synergy paradigm in seven CF ferrets. (A-G) Protocol and genotype are indicated on each time-MCC rate plot. Note that a reduced y-axis scale is used to display synergistic MCCV (E) and (G). Also note that the mean MCCV for T10-30 to 0.3 µM carbachol in (E) is less than 5% of that in (A) and (C). [Figure 8] Shows synergistic MCC with sequential agonists with methacholine and formoterol. [Figure 9] 1 shows the protective effect of formoterol against methacholine-induced muscle contractions. [Figure 10] Figure 1 shows how simultaneous treatment with formoterol and methacholine induces a synergistic response. [Figure 11] We show that simultaneous treatment with formoterol and methacholine does not induce muscle contractions. [Figure 12] Shown is the mean square displacement of particles transported in the ASL in response to treatment with DMSO control, forskolin, carbachol, forskolin + carbachol (SP), or the CFTR modulator combination elexacaftar (3 uM)-tezacaftar (3 uM)-ivacaftar (10 uM) (ETI) in cystic fibrosis and healthy adult human nasal epithelial cell cultures. [Figure 13] Figure 1 shows the effective diffusion ratio of mucus transport in cystic fibrosis and healthy adult human nasal epithelial cell cultures in response to treatment with DMSO control, forskolin, carbachol, forskolin + carbachol (SP), or the combination of elexacafter (3uM)-tezacafter (3uM)-ivacafter (10uM) (ETI). [Figure 14] 1 shows the effects of formoterol (Fmt) and Fmt plus methacholine (MCh) on TMV in a sheep cystic fibrosis model. [Figure 15] Figure 1 shows the effect of formoterol (Fmt) and Fmt + methacholine (MCh) on whole lung clearance in a sheep CF model. [Figure 16] 1 shows the effect of albuterol plus methacholine on the tolerability of single ascending doses in healthy volunteers. [Figure 17] Figure 1 shows the effect of formoterol plus methacholine on the tolerability of single ascending doses in healthy volunteers. [Figure 18] Figure 1 shows the effect of formoterol plus methacholine on the tolerability of single ascending doses in patients with CF. [Figure 19] Figure 1 shows sputum production (in grams) in CF patients in a single ascending dose tolerability study. [Figure 20] Figure 1 shows the percent solid content of sputum produced by CF patients in a single ascending dose tolerability study. [Figure 21] ENaC inhibition increases baseline MCCV but is not additive to dual agonist stimulation. Time course of MCCV in response to 10 μM benzamil (Bz, open red squares) or dual agonists in the presence (closed squares) and absence (open blue triangles) of benzamil harvested from WT newborn piglet tracheas (n=4 each). [Figure 22] Increased HCO3- secretion by synergistic agonists. Synergistic agonists significantly increased the rate of HCO3- secretion compared to baseline conditions (p=2E-05, n=13 from 8 pig tracheas). [Figure 23] Synergistic agonists increase ASL height in ex vivo WT pig tracheas. A. Change in mean ASL height with DMSO (open black squares, n = 2 pigs) or drugs (filled orange squares, n = 4 pigs). B. Summary of the rate of increase in ASL height at baseline (Bs), formoterol, and synergistic agonists. DETAILED DESCRIPTION OF THE INVENTION

[0011] definition Before describing the exemplary embodiments in more detail, the following definitions are set forth to illustrate and define the meaning and scope of terms used in the description.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those skilled in the art with the general meaning of many of the terms used herein. However, for clarity and ease of reference, certain terms are defined below.

[0013] Certain ranges are presented herein with the term "about" preceding the numerical values. The term "about" is used herein to literally support the exact numerical value that it precedes, as well as a numerical value that is close to or approximately the numerical value that it precedes. When determining whether a number is close to or approximately a specifically recited number, the close or approximate unrecited number may be a number that is substantially equivalent to the specifically recited number in the context in which it is presented.

[0014] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "β-adrenergic agonist" refers to one or more β-adrenergic agonists, i.e., a single β-adrenergic agonist and multiple β-adrenergic agonists. It should also be noted that the claims may be drafted to exclude any element. Accordingly, this statement is intended to serve as a prerequisite for using exclusive language, such as "only," "only," and the like, or for using "negative" limitations in connection with the recitation of claim elements.

[0015] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, the terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms "polynucleotide" and "nucleic acid," as applicable to the described embodiment, should be understood to include single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0016] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to polymeric forms of amino acids of any length, including coded and non-coded amino acids, amino acids that are chemically or biochemically modified or derivatized, and polypeptides with modified peptide backbones.

[0017] As used herein, the term "naturally-occurring" as applied to a nucleic acid, protein, cell, or organism refers to a nucleic acid, protein, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from nature and is present in an organism (including viruses) that has not been intentionally modified by humans in the laboratory is naturally-occurring.

[0018] The term "exogenous" as applied to a nucleic acid or protein herein refers to a nucleic acid or protein that is not normally or naturally found in and / or not produced by a particular bacterium, organism, or cell in nature. The term "endogenous nucleic acid" as used herein refers to a nucleic acid that is normally present in and / or produced by a particular bacterium, organism, or cell in nature. An "endogenous nucleic acid" is also referred to as a "native nucleic acid" or a nucleic acid that is "native" to a particular bacterium, organism, or cell. The term "endogenous polypeptide" as used herein refers to a polypeptide that is normally found in and / or produced by a particular bacterium, organism, or cell in nature.

[0019] As used herein, "recombinant" means that a particular nucleic acid or protein is the product of various combinations of cloning, restriction, and / or ligation steps, resulting in a construct with structural coding or non-coding sequences distinguishable from the endogenous nucleic acid found in natural systems. Typically, DNA sequences encoding structural coding sequences are assembled from cDNA fragments and short oligonucleotide linkers or from a series of synthetic oligonucleotides to provide a synthetic nucleic acid expressible from a recombinant transcription unit contained within an cellular or cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, i.e., introns, typically present in eukaryotic genes. Genomic DNA containing the relevant sequences can also be used to form recombinant genes or transcription units. Sequences of non-translated DNA can be present 5' or 3' from the open reading frame; these sequences do not interfere with the manipulation or expression of the coding region but rather serve to regulate the production of the desired product by various mechanisms.

[0020] Thus, for example, the terms "recombinant" nucleic acid or "recombinant" protein refer to a nucleic acid that is not naturally occurring but is instead created by the artificial combination, e.g., by human intervention, of two naturally separated segments of sequence. This artificial combination is often achieved by chemical synthesis means or by the artificial manipulation of isolated segments of nucleic acid, such as by genetic engineering techniques. This is usually done to replace codons with redundant codons that encode the same or a conservative amino acid, usually to introduce or delete sequence recognition sites. Alternatively, it is performed to combine nucleic acid segments of desired functions to produce a desired combination of functions. This artificial combination is often achieved by chemical synthesis means or by the artificial manipulation of isolated segments of nucleic acid, such as by genetic engineering techniques.

[0021] As used herein, the term "sample" refers to a substance or mixture of substances that is typically, but not necessarily, in liquid, i.e., aqueous, form and contains one or more components of interest. Samples can be obtained from a variety of sources, including food, environmental materials, biological samples, or solids such as tissues or bodily fluids isolated from an individual. This includes, for example, but is not limited to, plasma, serum, spinal fluid, semen, lymphatic fluid, external skin, respiratory, intestinal, urogenital, tear, saliva, milk, blood cells, tumors, organs, and samples of in vitro cell culture components (including, but not limited to, conditioned media obtained from growing cells in cell culture media, cells suspected of being infected with a virus, recombinant cells, and cellular components). In certain embodiments of this method, the sample includes cells. In some examples of this method, the cells are in vitro. In some examples of this method, the cells are in vivo.

[0022] The term "biological sample" includes clinical or non-clinical samples, including tissue obtained by surgical resection, tissue obtained by biopsy, cultured cells, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, etc. "Biological sample" includes samples obtained from sample cells of a patient, e.g., samples containing polynucleotides and / or polypeptides obtained from sample cells of a patient (e.g., cell lysates or other cell extracts containing polynucleotides and / or polypeptides), and samples containing sample cells from a patient. Biological samples containing sample cells from a patient may also include normal, non-diseased cells. Biological samples can be obtained from plants or animals. Biological samples can also be obtained from any species. In certain embodiments of the methods, the biological sample includes cells. In some examples of the methods, the cells are in vitro. In some examples of the methods, the cells are in vivo.

[0023] The term "antibody" encompasses: polyclonal and monoclonal antibody preparations, as well as preparations including hybrid, modified, chimeric, and humanized antibodies, and hybrid (chimeric) antibody molecules (see, e.g., Winter et al. (1991) Nature 349:293-299, and U.S. Pat. No. 4,816,567); F(ab')2 and F(ab)2 fragments; F vmolecules (non-covalent heterodimers, see, e.g., Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662, and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies (see, e.g., Hamers-Casterman et al. (1993) Nature 363:446; Desmyter et al. (2015) Curr. Opin. Struct. Biol. 32:1); dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327, Verhoeyan et al. (1988) Science 239:1534-1536, and GB Patent Application Publication No. 2,276,169, published September 21, 1994); and functional fragments derived from these molecules which retain the specific binding properties of the parent antibody molecule.

[0024] "Single-chain antibodies," "single-chain variable fragments," or "scFvs" comprise the heavy chain variable domain (VH) and light chain variable domain (VL) of an antibody connected by a flexible peptide linker, which is typically 10-25 amino acids in length. Single-chain antibodies retain the antigen-binding properties of natural full-length antibodies, but lack the Fc domain, making them smaller than natural intact antibodies or Fab fragments.

[0025] As used herein, the term "nanobody" (Nb) refers to the smallest antigen-binding fragment or single variable domain (V) derived from a naturally occurring heavy chain antibody. HH) and are known to those skilled in the art. They are derived from heavy chain-only antibodies found in camelids (Hamers-Casterman et al. (1993) Nature 363:446; Desmyter et al. (2015) Curr. Opin. Struct. Biol. 32:1). The family "Camelidae" contains immunoglobulins that lack light polypeptide chains. "Camelidae" includes Old World camelids (Camelus bactrianus and Camelus dromedarius) and New World camelids (e.g., Llama paccos, Llama glama, Llama guanicoe, Llama vicugna). Single variable domain heavy chain antibodies are referred to herein as nanobodies or V HH Nanobodies are smaller than human antibodies; nanobodies are typically 12-15 kDa, human antibodies are typically 150-160 kDa, Fab fragments are approximately 50 kDa, and single-chain variable fragments are approximately 25 kDa. Nanobodies offer certain advantages over conventional antibodies, including small size, ease of design, high chemical and thermal stability, good solubility, deep tissue penetration, the ability to bind to small cavities or difficult-to-access epitopes on target proteins, and the ability to be produced in microbial cells (i.e., lower production costs compared to animal immunization). As shown in McMahon et al. (2018) Nature Structural Molecular Biology 25(3): 289-296, which is incorporated herein by reference, specific nanobodies have been successfully generated using yeast surface display.

[0026] As used herein, "effective amount" or "therapeutically effective amount" refers to an amount of a compound of the present disclosure effective to achieve a desired therapeutic result, such as improving symptoms and / or reducing disease severity in an individual with cystic fibrosis, improving mucociliary clearance, increasing submucosal gland secretions, or inhibiting cholinergic agonist-induced muscle contractions. In the context of the present invention, a desired therapeutic result includes removing mucus from the lungs of such a patient or inhibiting the accumulation of mucus in the lungs of such a patient. The dosage amounts referred to in this disclosure are guidelines, but the attending physician can adjust the dosage depending on the specific needs of the patient, including, for example, the severity, size, and physical condition of the disease.

[0027] "Treating," "treatment," "preventing," "treating," "inhibiting," and corresponding terms include therapeutic treatment, prophylactic treatment, and treatment that reduces a subject's risk of developing a disease or risk factor. Treatment does not require a complete cure of the disease or condition, but includes disease-modifying effects such as reducing the severity, alleviating symptoms, reducing other risk factors associated with the condition, and / or slowing the progression of the disease.

[0028] Cystic fibrosis (CF) is a genetic disorder that primarily affects the gastrointestinal and respiratory systems, disrupting anion transport in exocrine glands and "wet" epithelia. It leads to chronic lung disease, exocrine pancreatic insufficiency, hepatobiliary disease, and abnormally high sweat electrolyte levels. Diagnosis is made by sweat testing or identification of two CF-causing gene mutations in patients with a positive newborn screening test or characteristic clinical features. Treatment is aggressive, multidisciplinary, and supportive care, along with small-molecule correctors and potentiators targeting defects in the cystic fibrosis transmembrane conductance regulator (CTFR) protein.

[0029] Cystic fibrosis is an autosomal recessive trait that affects approximately 3% of the Caucasian population, with a significantly lower incidence in non-Caucasian populations. The causative gene is known to be located on the long arm of chromosome 7, which encodes a membrane-associated protein called the cystic fibrosis transmembrane conductance regulator (CFTR). The most common genetic mutation, F508del, occurs in approximately 85% of CF alleles. Over 2,000 less common CFTR mutations have been identified.

[0030] CFTR is an anion channel controlled by cyclic adenosine monophosphate (cAMP). It conducts chloride and bicarbonate and influences the transport of other ions, particularly sodium, across epithelial membranes. It likely has many other functions. The disease manifests only in homozygotes. Heterozygotes exhibit subtle abnormalities in epithelial electrolyte transport that are largely inconsequential clinically but slightly increase the risk of many CF-related disorders (Miller, Proc Natl Acad Sci USA, 2020, 117, 1621).

[0031] CFTR variants are classified into six classes based on the variant's effect on the function or processing of the CFTR protein. Patients with class I, II, or III variants are considered to have a more severe genotype, resulting in little or no CFTR function, while patients with one or two class IV, V, or VI variants are considered to have a milder genotype, resulting in residual CFTR function. However, because there is no strict relationship between specific mutations and disease manifestations, clinical testing (e.g., tests of organ function) is more predictive of prognosis than genotyping. CFTR variants can include frameshift (deletions or insertions of DNA sequences, changing the way the sequence is read) or nonsense (stop) mutations.

[0032] Fifty percent of patients not diagnosed by newborn screening develop pulmonary symptoms, often during infancy. Pulmonary disease results from mucus blocking the airways. This mucus blockage promotes recurrent or chronic infections, with symptoms such as cough, phlegm production, and wheezing common. Cough is the most troublesome symptom and is often accompanied by phlegm, retching, vomiting, and sleep disturbances. As the disease progresses, symptoms such as intercostal retractions, accessory muscle use, barrel chest deformity, digital clubbing, cyanosis, and decreased exercise tolerance appear. Upper respiratory tract disease includes nasal polyps and chronic or recurrent sinusitis.

[0033] Beta-adrenergic agonists are drugs that relax airway muscles, widening the airways and easing breathing. They are a type of sympathomimetic drug, each acting on beta-adrenergic receptors. Generally, pure beta-adrenergic agonists have the opposite effect to beta-blockers. Beta-adrenergic receptor agonist ligands mimic the effects of both epinephrine and norepinephrine signaling in cardiac, pulmonary, and smooth muscle tissue. Epinephrine exhibits higher affinity. Activation of beta-1, beta-2, and beta-3 receptors activates the enzyme adenylate cyclase, which activates the second messenger cyclic adenosine monophosphate (cAMP). cAMP then activates protein kinase A (PKA), which phosphorylates target proteins, ultimately resulting in smooth muscle relaxation and cardiac contraction.

[0034] Cholinergic agonists are a class of medications that act on the neurotransmitter acetylcholine, the primary neurotransmitter within the parasympathetic nervous system (PNS). Cholinergic agonists stimulate cholinergic receptors, such as nicotinic and muscarinic receptors. There are two broad categories of cholinergic agonists: direct-acting and indirect-acting. Direct-acting cholinergic agonists act by directly binding to and activating muscarinic receptors. Examples of direct-acting cholinergic agonists include choline esters (acetylcholine, methacholine, carbachol, bethanechol) and alkaloids (muscarine, pilocarpine, cevimeline). Indirect-acting cholinergic agonists increase the availability of acetylcholine at cholinergic receptors. These include reversible agents (physostigmine, neostigmine, pyridostigmine, edrophonium, rivastigmine, donepezil, galantamine) and irreversible agents (echothiophate, parathion, malathion, diazinon, sarin, soman).

[0035] Detailed Description Before describing various embodiments, it is to be understood that the teachings of the present disclosure are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0036] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way. While the present teachings have been described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. Rather, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present teachings, some exemplary methods and materials are now described.

[0038] The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the claims are not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates that can be independently confirmed.

[0039] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features that may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present teachings. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0040] All patents and publications referred to herein, including all sequences disclosed within such patents and publications, are expressly incorporated by reference.

[0041] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that stated range, to the nearest tenth of the lower limit, is included in the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included in the invention.

[0042] It is understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments relating to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and expressly disclosed herein. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every subcombination were individually and expressly disclosed herein.

[0043] In further describing various aspects of the present invention, methods for treating individuals with cystic fibrosis are first described in detail. Methods for increasing submucosal gland secretion are then described. Methods for inhibiting cholinergic agonist-induced muscle contractions are then described. Finally, compositions for practicing the methods disclosed herein are described.

[0044] Methods of Treating Individuals with Mucosal Obstructive Diseases The present disclosure provides a method of treating an individual with a mucosal obstructive disease, the method comprising administering to the individual a β-adrenergic agonist or an adenylate cyclase activator in combination with a cholinergic agonist to treat the individual with the mucosal obstructive disease.

[0045] The methods disclosed herein can be used to treat a number of mucosal obstructive diseases, either directly treating the disease or alleviating symptoms associated with the disease. Mucosal obstructive diseases that can be treated by these methods include, but are not limited to, cystic fibrosis, primary ciliary motility disease, asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, chronic bronchitis, non-CF bronchiectasis, and the like.

[0046] If the mucosal obstructive disease is cystic fibrosis, the individual may be predicted to have or diagnosed with cystic fibrosis. Diagnosis may be based on a specific genetic mutation in the CFTR gene, resulting in a non-functional CFTR protein or reduced-functioning CFTR. Diagnosis may also be based on a symptom or collection of symptoms of clinical features. Genetic mutations associated with cystic fibrosis include, but are not limited to, G85E, R117H, 621+1G→T, 711+1G→T, 1078delT, R334W, R347P, A455E, ΔI507, ΔF508, 1717-1G-A, G542X, S549N, G551D, R553X, R560T, 1898+1G→A, 2184delA, 2789+5G→A, R1162X, 3659delC, 3849+10kbC, W1282X, and N1303K. Individuals may have other genetic mutations. The individuals for whom this therapy is used to treat cystic fibrosis and other mucosal obstructive diseases are typically mammals. Non-limiting examples of mammals that may be treated using the present methods include, but are not limited to, pigs, ferrets, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc. In some embodiments, the individual is a human.

[0047] The beta-adrenergic agonist of the present disclosure can be any beta-adrenergic agonist that activates beta-adrenergic receptors. The beta-adrenergic agonist can target any beta-adrenergic receptor that is considered useful, including beta-adrenergic receptors, beta-adrenergic receptors, or beta-adrenergic receptors. In some embodiments, the beta-adrenergic agonist is a beta-adrenergic agonist that targets beta-adrenergic receptors. Various β2-adrenergic agonists can be used to practice the methods disclosed herein, including, but not limited to, bitolterol, fenoterol, isoprenaline, isoproterenol, levosalbutamol, levalbuterol, orciprenaline, pirbuterol, procaterol, ritodrine, salbutamol, terbutaline, arformoterol, bambuterol, clenbuterol, formoterol, salmeterol, abediterol, carmoterol, indacaterol, olodaterol, vilanterol, isoxsuprine, mabuterol, zilpaterol, and the like. In some embodiments, the β2-adrenergic agonist is formoterol. In some embodiments, an adenylate cyclase activator, such as forskolin, is used instead of β2-adrenergic. Adenylate cyclase activators include forskolin and colforsin.

[0048] The cholinergic agonist of the present disclosure is any molecule that mimics the activity of the neurotransmitter acetylcholine and acts on muscarinic receptors. Various cholinergic agonists can be used in the methods embodied herein. The cholinergic agonist may be direct-acting or indirect-acting. Non-limiting examples of direct-acting cholinergic agonists include acetylcholine, methacholine, carbachol, bethanechol, muscarine, pilocarpine, and cevimeline. Non-limiting examples of indirect-acting cholinergic agonists include physostigmine, neostigmine, pyridostigmine, edrophonium, rivastigmine, donepezil, galantamine, echothiophate, parathion, malathion, diazinon, sarin, and soman. In some embodiments, the direct-acting cholinergic agonist is methacholine. In some embodiments, the direct-acting cholinergic agonist is carbachol.

[0049] The combination of a β-adrenergic agonist or adenylate cyclase activator and a cholinergic agonist can be administered at a specific time and / or in a specific order to treat individuals with cystic fibrosis or other mucosal obstructive diseases. Thus, the β-adrenergic agonist or adenylate cyclase activator and the cholinergic agonist can be administered sequentially or simultaneously. In some embodiments, the β-adrenergic agonist or adenylate cyclase activator is administered, followed by a delayed administration of the cholinergic agonist. In some embodiments, the cholinergic agonist is administered, followed by a delayed administration of the β-adrenergic agonist or adenylate cyclase activator. The delay can be of various durations. For example, the delay can be 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, 30 minutes or more, 35 minutes or more, 40 minutes or more, 45 minutes or more, 50 minutes or more, 55 minutes or more, 60 minutes or more, or more than about 60 minutes.

[0050] In some embodiments, a combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist is administered simultaneously. Simultaneous administration of a combination of a β-adrenergic agonist and a cholinergic agonist with an adenylate cyclase activator, or administration of a β-adrenergic agonist or an adenylate cyclase activator before a cholinergic agonist, may provide particular advantages. For example, administration of a β-adrenergic agonist or an adenylate cyclase activator simultaneously with or before administration of a cholinergic agonist can inhibit cholinergic agonist-induced airway smooth muscle contraction. Prior to the present disclosure, it was well established that a β-adrenergic agonist should not be administered before a cholinergic agonist. Unexpectedly, it has been found that administration of a β-adrenergic agonist before a cholinergic agonist can inhibit cholinergic agonist-induced airway smooth muscle contraction and airway narrowing. In some embodiments, administration of a β-adrenergic agonist or an adenylate cyclase activator in combination with a cholinergic agonist synergistically increases mucus transport compared to mucus transport with either agonist alone.

[0051] These methods include administering a β-adrenergic agonist or an adenylate cyclase activator in combination with a cholinergic agonist locally or systemically. When the combination is administered locally, it can be administered directly to the trachea or lungs or to a site near the trachea or lungs. When the combination is administered locally, it can be administered using an oral or nasal inhaler. When the combination is administered systemically, it can be administered in a convenient systemic form, such as a pill or oral tablet.

[0052] The combination of a β-adrenergic agonist or adenylate cyclase activator and a cholinergic agonist can be administered once daily, multiple times daily (e.g., twice or three times daily), intermittently, or weekly, depending on the dosage form (e.g., immediate-release or controlled-release) and individual needs. Administration can be long-term, intermittent, or limited, with repeated administration within the range determined by a qualified professional. For example, the combination can be administered daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or more than 30 days, and then discontinued. In some embodiments, the combination is administered intermittently, such as every 2 or 3 days, or every week.

[0053] In some embodiments, the method further comprises administering one or more cystic fibrosis transmembrane conductance regulator (CFTR) modulators. The combination of a β-adrenergic agonist or an adenylate cyclase activator with a cholinergic agonist has been shown to be effective in increasing mucus clearance when administered in combination with a CFTR modulator. When administered in combination with a combination of a β-adrenergic agonist or an adenylate cyclase activator with a cholinergic agonist, any CFTR modulator effective in treating cystic fibrosis can be used. CFTR modulators used in the present disclosure include, but are not limited to, elexacaftar, tezacaaftar, ivacaftar, lumacaftar, banzacaaftar, dutivacaaftar, and the like. In one embodiment, the one or more CFTR modulators are elexacaftar, tezacaaftar, and ivacaftar. In some embodiments, administering a combination of a β-adrenergic agonist or adenylate cyclase activator and a cholinergic agonist in combination with one or more CFTR modulators synergistically increases mucus transport compared to mucus transport of the combination alone or the one or more CFTR modulators alone.

[0054] In some embodiments, the combination of a β-adrenergic agonist or adenylate cyclase activator and a cholinergic agonist may be administered with other agents other than, or in addition to, one or more CFTR modulators. Other agents include agents that directly treat cystic fibrosis and other mucosal obstructive diseases or their symptoms, such as agents that improve mucus clearance. For example, other agents include, but are not limited to, recombinant human DNase, such as Pulmozyme, hypertonic saline, or powdered mannitol, as well as vectors designed to deliver agents, such as cDNA or mRNA, to airway cells designed to enhance defective CFTR protein.

[0055] Therapeutic methods disclosed herein may provide a variety of benefits to individuals with cystic fibrosis or other mucosal obstructive disorders. For example, administration may increase mucociliary clearance rate, increase airway submucosal gland secretion rate, and inhibit cholinergic agonist-induced airway smooth muscle contraction. Administration may also alleviate symptoms associated with cystic fibrosis or other mucosal obstructive disorders, including, but not limited to, recurrent or chronic infections, cough, sputum production, wheezing, abdominal distension, constipation, etc.

[0056] Method for increasing the rate of secretion of airway submucosal glands The present disclosure provides a method for increasing airway submucosal gland secretion in an individual, the method comprising administering to the individual a beta-adrenergic agonist or an adenylate cyclase activator in combination with a cholinergic agonist to increase the airway submucosal gland secretion rate in the individual.

[0057] The β-adrenergic agonist of the present disclosure can be any β-adrenergic agonist that activates β-adrenergic receptors. In one embodiment, the β-adrenergic agonist is a β2-adrenergic agonist that targets β2-adrenergic receptors. Various β2-adrenergic agonists can be used to practice the methods disclosed herein, including but not limited to: bitolterol, fenoterol, isoprenaline, isoproterenol, levosalbutamol, levalbuterol, orciprenaline, pirbuterol, procaterol, ritodrine, salbutamol, terbutaline, arformoterol, bambuterol, clenbuterol, formoterol, salmeterol, abediterol, carmoterol, indacaterol, olodaterol, vilanterol, isoxsuprine, mabuterol, zilpaterol, and the like. In some embodiments, the β2-adrenergic agonist is formoterol. In some embodiments, an adenylate cyclase activator, such as forskolin, is used in place of the β2-adrenergic agonist.

[0058] The cholinergic agonist of the present disclosure is any molecule that mimics the activity of the neurotransmitter acetylcholine and acts on muscarinic receptors. Cholinergic agonists can be direct-acting or indirect-acting. Non-limiting examples of direct-acting cholinergic agonists include acetylcholine, methacholine, carbachol, bethanechol, muscarine, pilocarpine, and cevimeline. Non-limiting examples of indirect-acting cholinergic agonists include physostigmine, neostigmine, pyridostigmine, edrophonium, rivastigmine, donepezil, galantamine, echothiophate, parathion, malathion, diazinon, sarin, soman, and the like. In some embodiments, the cholinergic agonist is methacholine. In some embodiments, the cholinergic agonist is carbachol.

[0059] The combination of a β-adrenergic agonist or adenylate cyclase activator and a cholinergic agonist can be administered at a specific time and / or in a specific order to treat an individual with cystic fibrosis. In some embodiments, the β-adrenergic agonist or adenylate cyclase activator is administered, followed by a delayed administration of the cholinergic agonist. In some embodiments, the combination of a β-adrenergic agonist or adenylate cyclase activator and a cholinergic agonist is administered simultaneously.

[0060] The methods disclosed herein increase the secretion rate of airway submucosal glands. The secretion rate of submucosal glands can be increased by a range of values. For example, administration of a combination of a β-adrenergic agonist and a cholinergic agonist can increase the submucosal gland secretion rate by at least about 0.5 nL / min, at least about 1.0 nL / min, at least about 1.5 nL / min, at least about 2 nL / min, at least about 3 nL / min, at least about 4 nL / min, at least about 5 nL / min, at least about 6 nL / min, at least about 7 nL / min, at least about 8 nL / min, at least about 9 nL / min, at least about 10 nL / min, or more than about 10 nL / min.

[0061] The secretion rate of submucosal glands can be increased in a variety of individuals. Individuals who may particularly benefit from such methods include those who have reduced mucus production, those who produce excessively viscous mucus, or those who suffer from chronic airway inflammation. Individuals who may respond to these methods are generally mammals. Non-limiting examples of mammals that can be treated using the present methods include, but are not limited to, pigs, ferrets, cows, goats, sheep, rodents, rats, mice, non-human primates, and humans. In some embodiments, the individual is a human. In some embodiments, the individual is a pig. In some embodiments, the individual is a ferret.

[0062] In some embodiments, the individual has a CFTR genetic variant, including, but not limited to, G85E, R117H, 621+1G→T, 711+1G→T, 1078delT, R334W, R347P, A455E, ΔI507, ΔF508, 1717-1G-A, G542X, S549N, G551D, R553X, R560T, 1898+1G→A, 2184delA, 2789+5G→A, R1162X, 3659delC, 3849+10kbC, W1282X, and N1303K. The individual may have other genetic mutations.

[0063] Method for inhibiting cholinergic agonist-induced airway smooth muscle contraction The present disclosure provides a method for inhibiting cholinergic agonist-induced airway smooth muscle contraction in an individual, the method comprising administering to the individual a β-adrenergic agonist or an adenylate cyclase activator, wherein the administration of the β-adrenergic agonist or adenylate cyclase activator occurs before or simultaneously with the administration of a cholinergic agonist to the individual to inhibit airway smooth muscle contraction.

[0064] The β-adrenergic agonist of the present disclosure can be any β-adrenergic agonist that activates β-adrenergic receptors. In one embodiment, the β-adrenergic agonist is a β2-adrenergic agonist that targets β2-adrenergic receptors. Various β2-adrenergic agonists can be used to practice the methods disclosed herein, including but not limited to: bitolterol, fenoterol, isoprenaline, isoproterenol, levosalbutamol, levalbuterol, orciprenaline, pirbuterol, procaterol, ritodrine, salbutamol, terbutaline, arformoterol, bambuterol, clenbuterol, formoterol, salmeterol, abediterol, carmoterol, indacaterol, olodaterol, vilanterol, isoxsuprine, mabuterol, zilpaterol, and the like. In some embodiments, the β2-adrenergic agonist is formoterol. In some embodiments, an adenylate cyclase activator, such as forskolin, is used in place of the β2-adrenergic agonist.

[0065] The cholinergic agonist of the present disclosure is any molecule that mimics the activity of the neurotransmitter acetylcholine and acts on muscarinic receptors. Cholinergic agonists can be direct-acting or indirect-acting. Non-limiting examples of direct-acting cholinergic agonists include acetylcholine, methacholine, carbachol, bethanechol, muscarine, pilocarpine, and cevimeline. Non-limiting examples of indirect-acting cholinergic agonists include physostigmine, neostigmine, pyridostigmine, edrophonium, rivastigmine, donepezil, galantamine, echothiophate, parathion, malathion, diazinon, sarin, and soman. In some embodiments, the cholinergic agonist is methacholine. In some embodiments, the cholinergic agonist is carbachol.

[0066] The methods disclosed herein inhibit cholinergic agonist-induced airway smooth muscle contraction. Cholinergic agonist-induced airway smooth muscle contraction can be inhibited by various amounts. For example, administration of a β-adrenergic agonist or an adenylate cyclase activator can inhibit cholinergic agonist-induced muscle contraction by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0067] Cholinergic agonist-induced airway smooth muscle contraction can be inhibited in various individuals. Individuals who have previously taken or regularly take cholinergic agonist-based drugs may particularly benefit from such methods. Cholinergic agonist-based drugs are used to treat various diseases, including, but not limited to, myasthenia gravis, xerostomia, urinary retention, neurogenic bladder, ophthalmic surgery support, glaucoma, dementia, acute colonic pseudo-obstruction, anticholinergic drug overdose, and Sjogren's syndrome. Individuals who respond to these methods are generally mammals. Non-limiting examples of mammals that can be treated using these methods include, but are not limited to, pigs, ferrets, cows, goats, sheep, rodents, rats, mice, non-human primates, and humans. In some embodiments, the individual is a human. In some embodiments, the individual is a pig. In some embodiments, the individual is a ferret.

[0068] In some embodiments, the individual has a genetic variant of CFTR, including, but not limited to, G85E, R117H, 621+1G→T, 711+1G→T, 1078delT, R334W, R347P, A455E, ΔI507, ΔF508, 1717-1G-A, G542X, S549N, G551D, R553X, R560T, 1898+1G→A, 2184delA, 2789+5G→A, R1162X, 3659delC, 3849+10kbC, W1282X, N1303K, and the like. The individual may have other genetic mutations.

[0069] composition The present disclosure also describes compositions for carrying out these methods. Generally, the compositions of the present invention may comprise a β-adrenergic agonist or adenylate cyclase activator, as described above, and a cholinergic agonist, in addition to pharmaceutical excipients as described above. The compositions may be contained in a device. In some embodiments, the device is an inhaler.

[0070] In some embodiments, the composition may further comprise one or more CFTR modulators. CFTR modulators of the present disclosure are described in more detail above. In some embodiments, the composition may further comprise other agents other than, or in addition to, one or more CFTR modulators. Other pharmaceutical agents include pharmaceutical agents that directly treat cystic fibrosis and other mucosal obstructive diseases or their symptoms, such as agents that improve mucus clearance. For example, other pharmaceutical agents include, but are not limited to, recombinant human DNase, such as Pulmozyme, hypertonic saline, or powdered mannitol, as well as vectors designed to deliver agents, such as cDNA or mRNA, to airway cells designed to enhance defective CFTR protein.

[0071] In some embodiments, the composition is formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate buffer, succinate buffer, citrate buffer, and phosphate buffer at concentrations ranging from 5 mM to 100 mM. In some embodiments, the aqueous buffer contains an agent that provides an isotonic solution. Such agents include, but are not limited to, sodium chloride and sugars (e.g., mannitol, dextrose, sucrose, etc.). In some embodiments, the aqueous buffer further contains a non-ionic surfactant, such as polysorbate 20 or 80. Optionally, the composition may further contain a preservative. Suitable preservatives include, but are not limited to, benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, etc. In most cases, the formulation is stored at about 4°C. Pharmaceutical compositions may also be lyophilized, which generally includes a cryoprotectant, such as sucrose, trehalose, lactose, maltose, or mannitol. Lyophilized formulations can be stored for extended periods at ambient temperatures.

[0072] Each of the active agents can be provided in a unit dose of about 0.1 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 50 μg, 100 μg, 500 μg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg, 500 mg, 750 mg or more.

[0073] The composition can be administered in unit dosage form and can be prepared by any method known in the art. Such methods include combining a β-adrenergic agonist or adenylate cyclase activator and a cholinergic agonist with a pharmaceutically acceptable excipient or diluent, which constitute one or more accessory ingredients. The pharmaceutically acceptable excipient is selected based on the chosen route of administration and standard pharmaceutical practice. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. The carrier may be solid or liquid, and the type is generally selected based on the type of administration to be used.

[0074] Examples of suitable solid carriers include lactose, sucrose, gelatin, agar, bulk powder, etc. Examples of suitable liquid carriers include water, pharmaceutically acceptable oils, fats, alcohols, or esters, emulsions, syrups, elixirs, suspensions, solutions and / or suspensions, and solutions and / or suspensions reconstituted from non-effervescent granules, as well as other organic solvents, including effervescent preparations reconstituted from effervescent granules. Such liquid carriers may contain, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners, melting agents, etc. Preferred carriers are edible oils, such as corn oil or canola oil. Polyethylene glycols, such as PEG, are also excellent carriers.

[0075] The combination of a β-adrenergic agonist or adenylate cyclase activator and a cholinergic agonist can be administered via pharmaceutical dosage forms known in the art, including, but not limited to, oral solids, oral liquids, injections, transdermal patches, and inhalants. Oral dosages are formulated for systemic administration, while inhaled dosages are formulated for local administration. Systemic formulations are intended to be digested in the stomach or intestines, while inhaled formulations are intended for direct local delivery to affected tissues, such as the trachea or lungs. Dosage forms may be formulated with excipients and other compounds to facilitate administration to a subject and maintain storage stability. See "Remington's Pharmaceutical Sciences" (Mack Publishing Co., Easton, PA). Oral pharmaceutical formulations include tablets, minitablets, pellets, granules, capsules, gels, liquids, syrups, and suspensions. This combination can be administered orally, typically as an oral solid, although an oral liquid may be preferable for certain patients, such as children and the elderly, who have difficulty swallowing tablets or capsules. Oral dosage forms may be immediate release or sustained release.

[0076] In one embodiment of the present invention, the combination of a β-adrenergic agonist or adenylate cyclase activator and a cholinergic agonist can be provided as an immediate-release formulation. The immediate-release combination can be administered once daily or in divided doses two, three, four, or more times daily. In another embodiment of the present invention, the combination is provided as a sustained-release formulation. A sustained-release formulation can provide patient convenience by reducing daily administration and improve patient compliance. Furthermore, the sustained-release formulation of the present invention can help reduce serum peaks and valleys, thereby reducing adverse events.

[0077] Oral controlled-release formulations are known in the art and include sustained-, extended-, delayed-, and pulsed-release formulations. See "Remington's Pharmaceutical Sciences" (Mack Publishing Co., Easton, PA). The active agent may be formulated in a matrix formulation comprising one or more polymers that slow the release of the drug from the dosage form, including hydrophilic or gelling agents, hydrophobic matrices, lipid or wax matrices, and biodegradable matrices. The active agent may be formulated, for example, in the form of beads having an inert sugar core and coated with known excipients to delay or slow the release of the active agent by diffusion. Enteric coatings are known in the art to be used to delay the release of an active agent until the dosage form transitions from the low pH environment of the stomach to the high pH environment of the small intestine, and can include methyl acrylate-methacrylic acid copolymer, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate (PVAP), shellac, sodium alginate, and cellulose acetate trimellitate.

[0078] The compositions of the present disclosure may be contained in a device. The device may be any device that allows the composition to be delivered to affected tissues, such as the trachea or lungs. Such devices include, but are not limited to, nebulizers, oral inhalers, nasal inhalers, etc. When the device is an inhaler, the inhaler can dispense a dry powder or liquid in the form of an aerosol spray. The aerosol spray is typically delivered from a pressurized pack using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, e.g., gelatin, for use in an inhaler can be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch. For example, a formulation for inhalation administration can be prepared according to the teachings of U.S. Patent No. 7,812,120 to Quay et al.

[0079] kit Kits for use in carrying out certain methods described herein are also provided. In certain embodiments, the kit includes, for example, a β-adrenergic agonist and a cholinergic agonist, as described above. In certain kits, the active agents are individually packaged or present in a formulation, for example, the active agents are delivered to a subject simultaneously. The active agents may be present in the same container or in separate containers, as appropriate.

[0080] In certain embodiments, the kits further include instructions for practicing the subject methods or means for obtaining same (e.g., a website URL that directs the user to a web page providing the instructions), which may be printed on a substrate, which may be one or more of a package insert, packaging, reagent containers, etc. Yet another form of these instructions is a computer-readable medium having information recorded thereon (e.g., a diskette, compact disc (CD), portable flash drive, USB storage, DVD, Blu-ray® disc, etc.). Yet another form of these instructions that may be present is a website address that can be used via the internet to access the information on the removed site.

[0081] Example The following examples are presented so as to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, such as bp (base pairs); kb (kilobases); pl (picoliters); s or sec (seconds); min (minutes); h or hr (hours); aa (amino acids); kb (kilobases); bp (base pairs); nt (nucleotides); im (intramuscular); ip (intraperitoneal); sc (subcutaneous), etc.

[0082] Example 1 Synergistic increase in MCCV in CF ferrets and WT pigs The "synergy paradigm" is a 2+The agonist group was defined as 30 min of continuous exposure to either a cAMP-inducing agonist, followed by at least 30 min of exposure to a combined agonist. 10 μM forskolin, isoproterenol, or formoterol were used as cAMP agonists, and 0.3 μM carbachol was used as a Ca agonist. 2+ were used as ascending agonists (all basolateral). In a previous study, their combination increased MCCV in ferret trachea to a level much greater than the predicted additive effect of the two agonists and approached a maximum ( 16 ).

[0083] To investigate whether a synergistic increase in MCCV occurs in the trachea of ​​CF ferrets, we examined the tracheas of seven transgenic adult CF ferrets of mixed genotypes (see the "Methods" section). The tracheas of seven CF ferrets were divided into two groups: four ferrets were first treated with forskolin, and three ferrets were first treated with carbachol, followed by the combined agonists. MCCV (all values ​​in mm / min) was then measured for 90–150 min and plotted as MCCV vs. time and agonist in Figure 1A. Without stimulation, MCCV decreased to nearly zero over the first 30 min (Figure 1A; see figure legend for details). Forskolin did not increase MCCV, whereas carbachol produced a slight increase. However, regardless of the order in which the agonists were combined, MCCV subsequently increased significantly and sustained, reaching approximately 20 mm / min. The mean data for the final 20 min of each basal and single-drug treatment period, and for the period from 10 to 80 min after the addition of the combined agonists, are shown as boxplots in Figure 1B. MCCV values ​​were 1.6 ± 1.09 (n = 3) for unstimulated, 0.18 ± 0.09 (n = 4) for 10 μM forskolin, and 3.29 ± 2.08 (n = 3) for 0.3 μM carbachol. "Total" is the arithmetic sum of the MCCVs induced by the two agonists used separately: 3.5 ± 2.07. SR, the synergistic response induced by the combined agonist, was 19.95 ± 4.12 (P = 0.006 for SR vs. Total).

[0084] Given these results, we asked whether synergy effects could be observed in different species. Intratracheal MCCV was measured from WT piglets aged 2–5 days (see the "Methods" section for details). Unstimulated MCCV velocity was less than 1 mm / min (mean T10–30 MCCV, 0.93 ± 0.39, n = 8 piglet tracheas), similar to that observed in WT ferrets (16). Eight piglet tracheas were divided into two groups: four were first treated with forskolin and four were first treated with carbachol, followed by the combined agonists. While carbachol or forskolin alone only slightly increased MCCV, the combined agonists produced a large and sustained increase in MCCV of 12–17 mm / min, regardless of the order of application (Figure 1C). The average data for the last 20 min of the basal and single agonist treatment periods and the last 50 min of the synergy paradigm period are shown as boxplots in Figure 1D. The mean MCCV values ​​were 0.3 μM carbachol: 0.91 ± 0.63 to 1.12 ± 0.82 (P = 0.32, Carb vs. Basal, 4 piglets), 10 μM forskolin: 0.95 ± 0.65 to 2.46 ± 0.68 (P = 0.13, n = 4), sum of individual responses (Sum): 3.58 ± 1.06, synergy paradigm (SR): 13.92 ± 0.94.

[0085] It is clear from the boxplots that the increase in MCCV in response to the combined agonist was significantly greater in the combined agonist than the arithmetic sum of the responses in both CF ferrets and WT pigs. Thus, the synergistic effect of MCCV exists in at least two species and persists, at least partially, after loss of CFTR function. The MCCV in CF ferrets under the synergistic condition was 5.7-fold faster than the arithmetic sum of the agonists used individually, but of practical importance is how this compares to WT ferrets. The CF ferret values ​​were compared to values ​​previously obtained from WT ferrets (16). With forskolin alone, the MCCV values ​​for WT and CF ferrets were 6.75 ± 0.84 (n = 28) vs. 0.18 ± 0.09 (n = 4). With carbachol alone, the MCCV values ​​for WT and CF ferrets were 8.24 ± 0.82 (n = 12) vs. 3.29 ± 2.08 (n = 3). For the synergistic responses to the combined agonists, the WT vs. CFMCCV values ​​were 36.24 ± 0.9 (n = 40) vs. 19.95 ± 4.12 (n = 7). Thus, compared with WT ferrets, CF ferrets responded approximately 0% to forskolin, approximately 40% to carbachol, and approximately 55% to synergistic responses.

[0086] In these experiments, forskolin was used to elevate cAMP. To evaluate clinically readily available β-adrenergic drugs, MCCV was measured in response to 10 μM of the β2-adrenergic receptor agonist formoterol instead of forskolin. In piglet tracheas, comparable synergistic increases in MCCV were observed (mm / min): baseline, 0.3 ± 0.1 (n = 12); 10 μM formoterol, 2.4 ± 0.9 (n = 5); 0.3 μM carbachol, 1.3 ± 0.8 (n = 3); and combined agonist, 10.9 ± 0.8 (n = 7) (Figure 1E, F).

[0087] The combined agonist did not induce airway smooth muscle contraction or airway narrowing. cAMP and Ca increase MCCV 2+ - Ca agonists also affect airway smooth muscle. When used alone, they have the opposite effect. 2+While cAMP-elevating agonists contract muscles, cAMP-elevating agonists relax muscles. When used therapeutically, the potential for unwanted bronchoconstriction by combined agonists poses a safety concern. To determine which effect predominates, we measured the airway smooth muscle responses to carbachol ± 10 mM forskolin or formoterol using two different methods: muscle tension and luminal area. We measured the tension of ferret tracheal muscle bundles in response to increasing concentrations of carbachol ± 10 mM forskolin. Forskolin inhibited the increase in tension up to 0.3 mM and 0.6 mM carbachol, significantly reducing the response to higher doses of carbachol (Figure 2A, B). Exposure to 0.3 mM carbachol resulted in a sustained 20–40% reduction in lumen area in thinly sliced ​​tracheal rings from piglets or ferrets, whereas administration of forskolin or formoterol prior to carbachol only resulted in a transient reduction of 5% or less (Figure 2C–F). Importantly, this protective effect was also observed in CF ferrets (Figure 2F).

[0088] The rate of mucus clearance reflects the transportability of mucus and the cilia beating frequency. Transportability is primarily determined by mucus moisture / concentration (17) and pH (or bicarbonate content) (18, 19). The primary source of upper airway fluid is the submucosal glands, and agonists used to stimulate MCCV also stimulate submucosal gland secretion (20-29). The depth and composition of the ASL also depend on the surface epithelium, which secretes and absorbs electrolytes / fluids. In fact, this is the primary means of controlling the ASL in airways lacking submucosal glands (30). Previous studies by us and others (26, 31-33) have demonstrated that cholinergic Na+ transport is essential for the regulation of ASL. + Evidence of an absorption inhibitor was found, suggesting a tendency to increase mucus fluidity and transport. Finally, CBF increases Ca 2+ (34) or cAMP (35). In the following experiments, we sought evidence to support or refute the possibility that each of these mechanisms contributes to the synergistic increase in MCCV.

[0089] Synergistic glandular mucus secretion in WT pigs, WT ferrets, and CF ferrets The synergistic increase in MCCV is partly dependent on increased mucus secretion from the submucosal glands. This hypothesis supports the idea that [Ca 2+ ] i Elevated agonists and [cAMP] i This comes from evidence that combinations of elevated agonists synergistically increase mucus secretion from submucosal glands in humans (22), pigs (24), and ferrets (21). However, those experiments used different specific concentrations of agonists. To determine whether the same protocol used here resulted in a synergistic increase in secretion from submucosal glands, mucus secretion rates of individual tracheal glands from WT pigs and ferrets, as well as from CF ferrets, were measured by time-lapse optical imaging (28) while stimulated with the same concentrations and exposure periods of agonists used in the MCCV studies. All secretion rates are reported as nanoliters / min / gland.

[0090] In WT pig tracheal glands, the mean unstimulated secretion rate was nl / min / gland (0.21 ± 0.06, 121 glands, 8 pigs, Figure 3A). Basal rates were significantly increased by each agonist alone and further increased when they were combined in either order. Rates to the combined agonist were significantly greater than the arithmetic sum of the individual responses: arithmetic sum = 1.26 ± 0.19 for 7 pigs vs. combined agonist = 2.86 ± 0.25 (2.3-fold greater, P < 0.01, 8 pigs). Data for individual pigs are shown in Figure 3B for those administered forskolin first and in Figure 3C for those administered carbachol first.

[0091] Similar results were obtained in WT ferrets (Figure 3D-F). In ferret tracheal glands, the mean rate of unstimulated secretion was approximately zero (0.003 ± 0.001, 67 glands, 7 ferrets, Figure 3D). This was significantly increased by forskolin (0.26 ± 0.07, 37 glands, 7 ferrets, P < 0.05) and carbachol (0.94 ± 0.28, 30 glands, 7 ferrets, P < 0.05). The secretion rate to the compound agonist was significantly greater than the arithmetic sum of the individual responses (Figure 3D): overall arithmetic sum = 1.27 ± 0.23 vs. 2.46 ± 0.39 for the compound agonist (1.9-fold greater, P < 0.05, 55-67 glands, 5-7 ferrets). Data for individual WT control ferrets are shown in Figure 3E when forskolin was administered first and in Figure 3F when carbachol was administered first.

[0092] Importantly, CF ferrets (CFTRKO / KO) also demonstrated synergistic glandular secretion despite not responding to forskolin alone. Only two CF ferrets were tested (Figure 3G-I). One CF ferret was first stimulated with 10 mM forskolin, and the other with 0.3 mM carbachol, both in a synergistic paradigm. Unstimulated secretion rates were near zero, similar to those in WT ferrets. Forskolin alone failed to stimulate secretion as expected (0.01, 7 glands). Carbachol alone increased the mean secretion rate to 0.45 ± 0.16, while the combined agonists increased the mean rates to 1.23 ± 0.35 (7 glands) and 1.31 ± 0.19 (7 glands). When the agonists were combined, synergistic effects were observed in both addition orders. The mean secretion rate for both ferrets to the combined agonist was 1.27 ± 0.15, which was 2.8 times the arithmetic sum of the two agonists used alone and approximately half the response of WT ferrets, 2.46 ± 0.39 (16).

[0093] To summarize this section, mucus secretion rates in both species, including CF ferrets, were increased to values ​​exceeding the additive values ​​of the drugs used alone, providing circumstantial evidence that glandular secretion rates contribute to MCCV in our system.

[0094] The combined agonist stimulates epithelial surface anion secretion and Na + inhibits the absorption of The surface epithelium also modifies the ASL. Figure 4A shows the major ion fluxes that control the depth of the ASL. Anion-mediated fluid secretion increases, while Na + Fluid absorption via the α-amyloid chain decreases ASL depth. It was hypothesized that combined agonists increase ASL depth and MCCV by stimulating secretion and inhibiting absorption (see also Figure 6). Figure 4B shows the best example of Isc traces from pig tracheal mucosa stimulated with forskolin followed by carbachol. Forskolin caused a sustained increase in Isc without a measurable change in conductance. Subsequent addition of 0.3 μM carbachol induced a transient increase in Isc (anion secretion), followed by a slow decrease in Isc and conductance, with conductance decreasing to 84% of its pre- and post-forskolin values ​​approximately 30 min later. The ENaC inhibitor benzamil (Bz) did not cause any further changes in Isc or conductance, indicating that carbachol inhibits ENaC-dependent Na+ secretion. + This suggests that absorption was completely inhibited. At this point, the epithelium secretes anions, as indicated by the rapid decrease in Isc and conductance produced by two anion channel inhibitors, BPO-27 and niflumic acid. In the absence of compensatory absorption, ASL depth would be predicted to increase unless MCCV increased (gold dotted line in Figure 6A). Our evidence indicates that MCCV increases.

[0095] Figures 4C–F show summary plots of ΔIsc as a function of time and stimulus. Each panel shows the response to 10 μM forskolin or 0.3 μM carbachol for the first 30 min, and the response to the combined agonists for the next 30 min, for WT pigs (Figure 4C, D) and WT ferrets (Figure 4E, F). Forskolin increased ΔIsc as expected in both species, but the addition of carbachol slowly decreased ΔIsc (Figure 4C, E). The interpretation of Isc in the forskolin + carbachol condition is that forskolin primarily stimulates anion secretion to increase Isc, while carbachol increases Na secretion. + It inhibited absorption and significantly reduced Isc. + Inhibiting absorption increases net liquid accumulation at the surface. When carbachol is first added, ΔIsc either decreases directly or transiently increases and then decreases (Figure 4D,F). In both cases, the subsequent ΔIsc increase for carbachol + forskolin is smaller than forskolin alone. This is because the effects on Isc are opposite, but the effects on ASL depth are additive.

[0096] Agonist stimulation of human cilia beat frequency (CBF) CBF is [Ca 2+ ] i (34) or [cAMP] iIt is known that CBF increases in response to an increase in either of the two agonists (35). To determine whether CBF exhibits a synergistic increase with the combined agonist, the CBF (Hz) of unstimulated human nasal cells in KRB (Krebs buffer solution) was 6.79 ± 1.69 at 25 °C and 10.46 ± 0.95 at 37 °C (four subjects, P = 0.01) (see the "Methods" section). As shown in Figure 5, neither agonist significantly increased CBF, but when combined, their additive effect increased CBF by 27.2% to 13.31 ± 0.77 Hz at 37 °C. This was a significant increase compared to unstimulated CBF (n = 4, P < 0.05), but not compared to the arithmetic sum of the ΔCBF for the two agonists: combined agonist: 2.85 ± 0.76, arithmetic sum: 2.19 ± 0.66 (n = 4, P = 0.47). Thus, although increases in CBF contribute to increases in MCCV, they are unlikely to explain the synergistic increases in MCCV seen with combined agonists (see “Discussion” section).

[0097] Consideration Key findings There were six major findings. (1) The combined agonist synergistically increased MCCV in transgenic ferrets with CF to 19.95 ± 4.12 mm / min, which is approximately 55% of the MCCV in WT ferrets tested under similar conditions. (2) The effect was not species-specific, as pigs also showed a synergistic increase in MCCV. (3) The combined agonist caused little or no airway constriction in WT ferrets, WT pigs, and transgenic ferrets with CF. Potential mechanisms include: (4) a synergistic increase in glandular mucus secretion in pigs, ferrets, and CF ferrets; (5) increased anion secretion by the surface epithelium; and (6) increased Na secretion by the surface epithelium. +(7) Absorption was decreased, and CBF was increased. The magnitude of the synergistic increase in MCCV was several-fold higher than either agonist alone or their combined responses and approached the maximum reported in vivo. In anesthetized ferrets, the basal MCCV in vivo was 18.2 ± 1.0 mm / min, which increased to 32.0 ± 3.8 mm / min with maximal anticholinesterase treatment (36). In anesthetized pigs, the mean basal MCCV in vivo was approximately 7 mm / min, and the mean maximum MCCV was approximately 12 mm / min (37). If a synergistic effect also occurs in vivo, it should aid mucus movement in certain obstructive airway diseases.

[0098] The most notable result was a synergistic increase in MCCV in CF ferrets, which has therapeutic implications and is mechanistically intriguing because forskolin alone did not increase MCCV (Figure 1A) or stimulate glandular mucus secretion (Figure 3H) in the trachea of ​​CF ferrets. (Using a different synergy paradigm, human submucosal gland secretion was abolished in the airways of CF patients (22)).

[0099] Therefore, the combined agonists used in this study must be activating a CFTR-independent anion secretion pathway that is insensitive to forskolin alone (see below).

[0100] Strategies to enhance mucus clearance Strategies to enhance mucus clearance are the mainstay of cystic fibrosis treatment, but their effectiveness is modest (6, 8-10). Pulmozyme (recombinant human DNase), hypertonic saline, and mannitol all improved mucus clearance in cystic fibrosis, and inhaled bicarbonate or tromethamine improved sputum rheology in cystic fibrosis (38).

[0101] Long before treatment with Pulmozyme or hypertonic saline, numerous studies have demonstrated that β-adrenergic (cAMP) agonists increase MCC (13, 39). Indeed, β-adrenergic agonists, considered bronchodilators, are now widely used to treat obstructive diseases. However, the doses required to induce an increase in MCC are higher than those that reliably produce bronchodilation (39), so it is unclear to what extent currently used doses increase MCC. Unlike β-adrenergic agonists, cholinergic agonists (Ca 2+ ) causes bronchoconstriction, which is the basis for the methacholine challenge test (40), and cholinergic stimulation has been reported to increase mucus transport in humans (41). Cholinergic agonists also stimulate mucus secretion, and excessive mucus production is widely believed to contribute to mucus-obstructive disorders (42, 43). Therefore, it is not surprising that no one has previously proposed the therapeutic use of inhaled agents that stimulate mucus secretion and cause bronchoconstriction. Indeed, anticholinergic drugs are used to treat COPD, and their modest effects are thought to be primarily due to increased bronchodilator activity (44). Therefore, our finding that the combination of forskolin (or the β-adrenergic agonist formoterol) with a low dose of a cholinergic agonist significantly increases MCCV was unexpected.

[0102] Our hypothesis is that the primary reason combined agonists increase MCCV is because they increase ASL volume through three processes: a synergistic increase in gland mucus secretion, increased fluid secretion, and decreased absorption by the surface epithelium (Figure 6). Combined agonists produced only a small additive increase in CBF measured with Krebs solution. The larger increase in CBF may be dependent on the increased ASL volume, which occurred in the MCCV but not the CBF experiments. Using micro-optical coherence tomography (μOCT) to visualize transport in intact tracheas, an increase in CBF in response to cholinergic stimulation has been observed (45, 46). Importantly, all of this occurs in the absence of airway constriction.

[0103] The idea that increased ASL depth results in faster MCCV is supported by studies of patients with pseudohypoaldosteronism (PHA), in which loss-of-function mutations in ENaC subunits eliminate Na absorption from the airway surface, resulting in a more than two-fold increase in ASL volume and a four-fold increase in the clearance rate of inhaled tracers from the lung between 0 and 20 min (47). Previously, it was demonstrated that agonist-induced MCCV in ferrets approximately doubles when ENaC is inhibited (16). In those experiments (16), stimulation with either forskolin or carbachol in the presence of ENaC inhibition increased MCCV to a similar level to that seen with combined agonists, providing further evidence that synergistic MCCV results in part from ENaC inhibition. The idea that increased ASL results in faster clearance also underlies the logic behind the use of β-agonists (39) and hypertonic saline (8, 9) to enhance clearance. This is supported by studies of porcine tracheas in vitro, where stimulating secretion increased MCCV, inhibiting secretion slowed MCCV, and inhibiting absorption increased tracheal MCCV after secretion was inhibited ( 20 ).

[0104] Potential molecular and cellular mechanisms Although the molecular and cellular mechanisms responsible for the synergistic MCCV by β-adrenergic and cholinergic agonists were not addressed in this study, previous studies on the molecular mechanisms of ENaC inhibition are relevant given the evidence that ENaC inhibition contributes. A common theme is [Ca 2+ ] i (48-50) This can be achieved by a wide range of agonists, including ATP, UTP, histamine, thapsigargin, and bradykinin (51). Cholinergic agonists increase [Ca 2+ ] i Other mechanisms include increased extracellular antiproteases (27, 52) and other ENaC inhibitors, which stimulate secretions from airway glands and surface epithelia (25, 53).

[0105] A synergistic increase in MCCV and glandular secretion was observed in CF ferrets. Therefore, a mechanism bypassing CFTR must be involved in part. It is believed that the inositol 1,4,5-trisphosphate receptor-binding protein (IRBIT) pathway increases cAMP and Ca2+ release. 2+ Intracellular crosstalk between signaling pathways has been shown to mediate synergistic effects in salivary glands and pancreatic ducts (54). The synergistic secretion of lacrimal glands in response to cAMP and cholinergic agonists was partially attributed to the inhibition of p44 / p42 mitogen-activated protein kinase (MAPK) by cAMP agonists (55). In a previous study (50), cAMP + Ca was observed in serous cells isolated from human nasal glands and tracheal glands of wild-type and CFTR- / - pigs. 2+ Agonist-induced synergistic fluid secretion is due to Ca 2+ Agonist plus cAMP-dependent Ca 2+ Ca release mechanism 2+ However, another study using HEK293 cells (49) demonstrated that cAMP agonists such as parathyroid hormone and isoproterenol alone did not induce [Ca 2+ ] i However, when combined with carbachol, cAMP agonists increase the individual Ca2+ receptors in the ER. 2+ Exposing the carbachol-induced Ca pool 2+ The discrepancies in previous reports are due in part to the use of different cell or organ preparations and in part to different measurement parameters (e.g., [Ca 2+ ] i and [HCO3 - ] i ([pH] i Our previous studies (22, 24) have demonstrated that there are CFTR-dependent and -independent pathways for synergistic glandular mucus secretion depending on the dose of β-adrenergic and cholinergic agonists.

[0106] Potential therapeutic relevance for mucosal obstructive diseasePatients with mucosal obstructive airway diseases require treatments to enhance mucociliary clearance, including a significant number of CF patients (11, 56). Because β-agonists and methacholine are routinely used (the latter to test for overactive airways), there should be little difficulty in testing them in combination, even if it seems counterintuitive. Our ex vivo data demonstrate that this combination is effective in speeding mucus clearance without causing airway narrowing, even in CF animals with airway muscles that are more sensitive to cholinergic agonists (Figure 2) (57). Our results regarding CF airways are consistent with a previous study that observed a significant reduction in bronchoconstriction when CF children were given a β-adrenergic agonist before methacholine (58).

[0107] It remains unclear whether this combination is safe for individuals with overactive airways. If the results warrant further trials in CF patients, early initiation in healthier airways will be important, as a trend toward greater benefit in healthier airways compared with diseased airways was observed for MCC improvement with β-agonists (39).

[0108] Materials and Methods Airway tissue procurement CF ferret tissues. Seven transgenic CF ferret tracheae (CFTR G551D / G551D 5. CFTR ΔF508 / ΔF508 One, CFTR G551D / KO One CFTRKO / KO ferret trachea was used for the MCC assay. These ferrets were raised on the CFTR modulator VX770. Treatment was discontinued at least 3 weeks before euthanasia. No residual drug effects were expected or observed (zero response to forskolin). Two CFTRKO / KO ferret tracheas were used for the tracheal single gland mucus secretion rate assay. Two CF ferret tracheas (one CFTRKO / KO) were used for the tracheal smooth muscle contraction assay. ΔF508 / ΔF508 , and the other is CFTR G551D / ΔF508All isolated CF ferret tracheal trims (2-3 cm in length) were placed in DMEM medium immediately after euthanasia and shipped via next-day priority express from the University of Iowa.

[0109] Pig tissues. Newborn WT piglet tracheas (2–5 days old) were obtained directly from the University of California, Davis, pig facility or via next-day priority express from David Stoltz's laboratory at the University of Iowa. Postmortem (less than 1 hour) tracheas from young adult Yorkshire pigs (30–50 kg) and 5–12-month-old M. putorius ferrets were obtained from Stanford University and its animal facility in Gilroy, California. All procedures using animal tracheas were performed in accordance with relevant guidelines and regulations at Stanford University, and the animal protocol was approved (Stanford IACUC protocol number: 10,048). Piglet tracheas were transported in DMEM cell culture medium, while other animal tissues were transported to the laboratory in cold PhysioSol™ solution (Hospira, IL, USA) and then transferred to ice-cold Krebs Ringer bicarbonate (KRB) buffer gassed with 95% O2 and 5% CO2 and stored at 4 °C until use. The KRB buffer contains 115 mM NaCl, 25 mM NaHCO3, 2.4 mM K2HPO4, 0.4 mM KH2PO4, 1.2 mM MgCl2, 1.2 mM CaCl2, 10 mM glucose, and 1.0 μM indomethacin, adjusted to pH 7.2 and approximately 290 mOsm at room temperature.

[0110] Human tissue. Human nasal mucosal tissue was collected from nasal biopsies during endoscopic sinus surgery at Yonsei University Hospital. All methods using human tissue were carried out in accordance with the relevant guidelines and regulations of Yonsei University, Seoul, Korea. All experimental protocols were approved by Yonsei University, and informed consent was obtained from all participants prior to the study (Yonsei University-IRB protocol number: 4-2016-1153).

[0111] MCCV measurementDetails can be found in previous reports (16, 59, 60). Briefly, the entire length of a ferret or piglet trachea, or a CF tracheal trim, was dissected along the dorsal midline and mounted mucosally side up on a Sylgard elastomer platform. For pig experiments, newborn piglet tracheas were used. This was because the tracheas of adult pigs undergoing acute experiments, the source of submucosal gland experiments, exhibited irregular MCC rates due to epithelial damage caused by intubation. The prepared trachea, with the serosal surface immersed in KRB buffer solution ± drug, was placed in a sealed, humidified chamber continuously insufflated with gas (95% / 5% O2 / CO2). During the initial 30-minute stabilization period, the tissue was immersed in a bath, and the temperature was gradually raised to 37°C. Afterwards, excess apical solution was drained, and the tissue was incubated for an additional 10 minutes before starting baseline MCCV measurements. Drugs were added by replacing the drug with a pre-warmed bath. Summary MCCV data are reported as a single number based on the average MCCV during the last 20 minutes of the treatment period unless otherwise stated.

[0112] electrophysiology Ferret tracheal section (approximately 0.5 × 1.0 cm) 2 ) or cartilage, mounted in an EasyMount Ussing chamber (Physiologic Instruments, California, USA) with an exposed surface area of ​​0.45 cm, immersed in KRB buffer at 37°C, and maintained in 95% O 2 / 5%CO 2 The tissue was gassed with 0.05% CO₂. Transepithelial short-circuit current (Isc) was acquired and displayed using a VCC-600 voltage clamp (Physiologic Instruments, CA, USA) and PowerLab Chart4 software (v.4.1.2, https: / / adinstruments.com, ADInstruments, CO, USA). Total tissue conductance was calculated by applying Ohm's law to the Isc deflection produced by applying a 1 mV pulse across the tissue every 20 seconds during the experiment. Unless otherwise stated, the average response was reported for the last 20 minutes of each measurement period.

[0113] Optical measurement of gland mucus secretion rate Details are given in a previous report (28). Ferret tracheal sections (approximately 1.5 cm) were cut free from the underlying cartilage in cold Krebs Ringer bicarbonate buffer. 2 ) or porcine tracheal mucosa was placed, mucosal side up, on a flexible silicone-lined 35-mm Petri dish. The surface was dried and then covered with water-saturated mineral oil while the glands were immersed in KRB buffer. The appearance of "mucus bubbles" within the oil layer was visualized by oblique illumination, and digital images were taken with the macro lens of a Nikon digital camera. Stored images were analyzed by direct measurement or using ImageJ software (v.1.50i, https: / / imagej.nih.gov / ij / , NIH, MD, USA). The indicated drug rates were calculated over 5-min intervals based on the average of the sustained T10-30 secretion rate induced by 10 mM forskolin or 0.3 mM carbachol alone, or the T5-30 secretion rate induced by the combined agonist. Rapidly fusing bubbles induced by the combined agonist were also included.

[0114] Ciliary beat frequency measurementIn laboratories where ferret or pig tracheal mucosa is not readily accessible, cilia beating frequency was measured using human nasal mucosa. Human nasal mucosa collected from endoscopic nasal biopsies was further dissected under a microscope and placed in a temperature- and pH-controlled chamber. Perfused Krebs bicarbonate buffer was maintained at 37°C and pH 7.4. Cilia were visualized using differential interference contrast (DIC) optics with a Zeiss microscope (Munich, Germany) equipped with a 40x or 60x objective. Images were displayed live and automatically captured at 2,000 fps using a high-frame-rate digital camera (optiMOS and NIS-Elements microscope imaging software (Nikon, Japan)) and converted to TIFF images. Images were acquired for 10 seconds for each condition, and experiments were performed in the following order: (1) unstimulated CBF at room temperature; (2) unstimulated CBF at 37°C; (3) CBF at 37°C with 0.3 μM carbachol; (4) 10-minute washout; (5) CBF with 10 μM forskolin; and (6) CBF with 0.3 μM carbachol plus forskolin. Each condition was maintained for at least 10 minutes. Note that this paradigm differs from the synergy paradigm used to measure MCCV and glandular mucus secretion rate in that the exposure to agonist was 10 minutes rather than 30 minutes, and the condition in which forskolin was added in addition to carbachol was omitted. All recordings for each condition were made in three different regions of the epithelium, and the analyzed CBF was averaged for each experiment. In-house coding using MATLAB® software (MA, USA) was used to analyze the captured images and calculate CBF.

[0115] Airway smooth muscle contraction measurementTwo methods were used to measure tracheal smooth muscle contraction. One method is designed to measure airway narrowing using thinly sliced ​​tracheal rings. Piglet or ferret tracheal ring specimens (approximately 2 mm) were submerged and firmly fixed in a Sylgard-lined Petri dish filled with KRB solution at 37°C, pH 7.4. Digital images of tracheal ring contractions in response to agonist stimulation were recorded with a Nikon digital camera at 1- to 10-minute intervals, and the luminal surface area of ​​the tracheal rings was calculated using ImageJ (NIH, MD, USA). The other method uses a force transducer. One end of an isolated ferret tracheal muscle bundle was fixed in a Sylgard-lined Petri dish filled with KRB solution, and the other end was connected to a pre-calibrated strain gauge (Series 400A Force Transducer System, Cambridge Technology, MA, USA) with 26-gauge wire. Tension responses to increasing doses of carbachol ± 10 μM forskolin were obtained and displayed with PowerLab Chart4 software (ADInstruments, Colorado, USA).

[0116] reagent Chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), Calbiochem (Billerica, MA, USA), and Alomone Labs (Jerusalem, Israel). BPO-27 was provided by Alan Verkman at UCSF. Forskolin, benzamil, BPO-27, niflumic acid, and formoterol fumarate were dissolved in dimethyl sulfoxide (DMSO), carbachol was dissolved in sterile double-distilled water, and indomethacin was dissolved in absolute ethanol. Solutions were either made fresh or stored at -20°C as aliquots of stock concentration. All chemicals were diluted 1:1000 in Bass KRB solution immediately before use (except indomethacin, which was diluted 1:10,000) and used at the concentrations indicated.

[0117] statisticsData are presented as mean ± SEM unless otherwise noted. Student's paired and unpaired t-tests or Mann-Whitney U test were used to compare means of different treatment groups.

[0118] Example 2 Effects of sequential and simultaneous administration of formoterol and methacholine on mucociliary clearance rate To investigate whether methacholine and formoterol induce a synergistic increase in MCCV, piglet tracheas were treated with 0.3 μM methacholine followed by 10 μM formoterol, or 10 μM formoterol followed by 0.3 μM methacholine. MCCV (all values ​​in mm / min) was then measured for 90–120 min and plotted as MCCV vs. time and agonist in Figure 8. MCCV decreased to nearly zero during the first 30 min without stimulation (Figure 8). Formoterol did not increase MCCV, and methacholine only slightly increased it. However, subsequent administration of the agonists, in either order, resulted in a large and sustained increase in MCCV, reaching approximately 12 mm / min. This synergistic increase in MCCV was also observed when methacholine and formoterol were administered simultaneously (Figure 10).

[0119] Effects of sequential and simultaneous administration of formoterol and methacholine on airway smooth muscle contraction To investigate whether formoterol can also inhibit cholinergic agonist-induced airway smooth muscle contraction, we measured the response of airway smooth muscle to methacholine (0.3 or 0.6 μM) ± 10 mM formoterol by measuring luminal area. Lumen area imaged in thinly sliced ​​piglet tracheal rings showed a sustained 20–40% reduction following exposure to 0.3 or 0.6 mM methacholine, whereas administration of formoterol prior to methacholine only caused a transient reduction of 5–10% (Figure 9). Coadministration of methacholine and formoterol also demonstrated a protective effect against methacholine-induced airway smooth muscle contraction (Figure 11).

[0120] Effects on mucus transport at the epithelial level in an in vitro cell culture model Human nasal epithelial cells (HNECs) were collected from CF patients (F508 del homozygotes) and healthy WT controls according to established standard operating procedures. Cells were then dissociated and seeded onto collagen-coated 0.4 μm pore polyester membrane inserts (Corning Inc.), grown in Pneumacult Ex-Plus medium (StemCell Technologies), and added to both the basal and apical chambers. The apical medium was then removed to create an air-liquid interface (ALI), and the basal medium was replaced with Pneumacult ALI medium (StemCell Technologies). After HNE CALI cultures matured and demonstrated the presence of active cilia, CF cells were treated with DMSO control or a combination of elexacafter (3 μM), tezacafter (3 μM), and ivacafter (10 μM) (ETI) for 48 hours. After 48 h, cells were treated with DMSO control, forskolin, carbachol, or forskolin plus carbachol (SP), inserts were excised from the support, placed in medium-filled concave-well slides so that only the basal side was exposed to the medium, and 20 μL of a 0.1% suspension of 2 μm fluorescent polystyrene beads (Thermo Scientific R0200) was added to the apical surface.

[0121] The slides were then placed on a custom-built system containing a stage heated to 37 °C and photographed from above with a digital microscope equipped with a high-speed camera (Keyence Inc., Elmwood Park, NJ). Images were acquired at a frame rate of 1000 fps and exported to ImageJ to analyze particle movement using the MTrackJ plugin (v.1.5.1). Wild-type cells were used as a reference control. The extracted frame-by-frame coordinates were then used to estimate the distance traveled by individual particles. The coordinates and time difference between frames were used to define the time scale (τ) according to the multiple particle transport (MPT) methodology, and the mean squared displacement (MSD) was then determined as the squared displacement of the particle over all possible time differences. The effective diffusivity (D) was then calculated from the extracted MSD and time scale. eff) were calculated. The MSD and D eff Data were analyzed by Loess smooth regression and MSD and D were calculated with 95% confidence intervals to allow comparisons between treatment conditions. eff Ensemble plots of time scale were generated (Figs. 12 and 13).

[0122] Example 3 Inhaled administration of a combined agonist significantly improves MCC in sheep with induced CF airway disease. To determine whether the synergistic increase in MCCV observed in the in vitro and in vivo experiments described above could be generated in vivo in a CF animal model, studies were conducted in adult sheep. In this established CF model (Abraham, W. Pulm Pharm Ther 2008; Kim et al.; Am J Respir Crit Care Med 2020, 201:313-324), a CF-like state is created in the airways of adult sheep by aerosolization of CFTRinh172 followed by human neutrophil elastase. As a large animal model, it provides an opportunity to evaluate the effects on the trachea, which has characteristics similar to those in CF patients, and nearly directly reflects inhaled dose studies. Briefly, adult sheep are positioned upright in a specialized body harness and, under local anesthesia, a standard endotracheal (ET) tube is inserted through the nose. Tracheal mucus velocity (TMV), the in vivo equivalent of MCCV, is measured by fluoroscopy. Five to seven radiopaque Teflon / bismuth trioxide disks (1 mm diameter, 0.8 mm thickness, 1.8 mg weight) are injected into the central part of the animal's trachea. As the disks are delivered into the trachea, the cranial velocity of each disk is recorded on videotape from a portable image intensifier connected in series with the fluoroscopy device. The average value of all disk velocities at a given time point is calculated, and a new disk is insufflated at subsequent time points. After a baseline value is determined, the animal is administered 10 mg of CFTRinh172 by aerosolization, and TMV measurements are repeated hourly for the next 2 hours. The animal is then administered 1190 mU of human neutrophil elastase (hNE) by aerosolization, and TMV measurements are repeated hourly for the next 2 hours. This induces a significant and persistent TMV disorder typical of CF. Once CF conditions were established, animals received either vehicle control (n = 4), 20 μg formoterol (n = 2), or a combination of 20 μg formoterol and 12 μg methacholine (n = 2) by inhalation. Methacholine alone was not tested because it could cause severe bronchospasm and endanger the animals' lives. After inhalation test drug administration, TMV was measured every 12 hours and at 24 hours.As shown in Figure 14, after TMV suppression with CFTRinh172+hNE, formoterol alone had little effect on TMV compared to controls, while the synergistic agonist increased TMV to approximately 80% of the pre-suppression baseline. Importantly, the effect persisted for 24 hours after a single dose. Because the TMV study only assessed intratracheal MCC and our ultimate goal was to eliminate mucus accumulation in the lungs, the TMV study was complemented by a whole-lung clearance study. To accomplish this, the same CF sheep model was used, but whole-lung MCC was assessed by radionuclide clearance techniques. For this, after administration of CFTRinh172 and hNE, 99mTc sulfur colloid was administered by inhalation, followed by either control vehicle or a combination of 20 μg formoterol and 12 μg methacholine. Radiotracer activity in the lungs was then monitored over a two-hour period with a gamma camera, as previously described (Abraham, Pulm Pharmacol Ther 2008, 21, 743-75). As shown in Figure 15, in control conditions (n=2), the radiotracer was retained over the two-hour observation period, giving the artifactual appearance of increased signal, while in synergistically treated animals (n=2), radiotracer activity was reduced by 11%. Of particular note was the lack of adverse effects observed in sheep exposed to the synergistic combination.

[0123] Administered as a single dose by inhalation, the combined agonist is well tolerated by healthy human volunteers and CF patients. Given these experimental results, we desired to evaluate whether the combination of an inhaled β-adrenergic agonist and a cholinergic agonist could be tolerated in humans. This would establish a basis for proceeding with the full development of the therapeutic agent for clinical application. Three ascending-dose single-dose studies were conducted in human volunteers and CF patients. These studies used available clinical-grade agents. The first study aimed to test the effects of combining albuterol, a short-acting β-adrenergic agent, with methacholine at 0, 1, 3, or 12 μg in three dose cohorts of healthy volunteers. Overall, all doses tested were well tolerated, no adverse effects were observed, and the predefined safety endpoint of no subjects experiencing a 20-point or greater decline in forced expiratory volume in 1 second (FEV1, a standard measure of airway obstruction) was met (Figure 16). This study was followed by a similar study, but this time using formoterol, a long-acting β-adrenergic agent, with which most of our experimental work was conducted. This study was similar to the first, in which human volunteers in cohorts of three subjects received a combination of inhaled formoterol and 0, 1, 3, or 12 μg of methacholine. Again, no signs of intolerance were observed at any of the doses tested, and the study met its predefined safety endpoint, this time a more stringent one: a 10-point or greater decline in FEV1 (Figure 17). Following these promising results, a study was conducted in CF patients. This study was similar to the healthy volunteer study using formoterol and methacholine, with the only difference being that the dosing cohort size was increased to six subjects. As with the previous study, no adverse effects were observed at any of the doses tested, and this study also met the safety endpoint of no 10-point or greater decline in FEV1 (Figure 18). As an exploratory endpoint in the CF study, sputum production by subjects was assessed as a reflection of the effect on mucus clearance from the lungs.We observed a greater increase in sputum volume (g, Figure 19) with the highest methacholine dose, but this did not reach statistical significance (p=0.056) due to the small sample size of each treatment cohort. However, we did find a statistically significant increase in sputum solids content in the methacholine-treated group (p=0.024, Figure 20). This is interpreted as reflecting the displacement of denser material, characteristic of residual secretions in the CF lung.

[0124] Example 4 Further evidence that inhibition of ENaC-mediated transport is a component of the synergistic increase in MCCV. Previous studies have demonstrated that stimulation of ferret tracheal tissue with forskolin or carbachol in the presence of ENaC inhibitors increases MCCV values ​​similar to those observed with combined agonists, suggesting that ENaC inhibition may be involved in the synergistic MCC induced by combined agonists. It was hypothesized that ENaC inhibition may be involved in the synergistic MCC induced by β-adrenergic and cholinergic agonists. Consistent with this hypothesis, MCCV results induced by 10 μM formoterol plus 0.3 μM methacholine were comparable in the presence (mm / min) of ENaC inhibition (10 μM benzamil) and absence (0.1% DMSO as control treatment): 14.0 ± 1.6 with benzamil and 13.9 ± 1.6 without benzamil (P = 0.97, n = 4 piglets). As shown in Figure 21, baseline MCCV was significantly increased by almost 6-fold by ENaC inhibition: 3.0±0.7 with benzamil and 0.5±0.7 without benzamil (P=0.02, 4 piglets).

[0125] ASL pH may affect mucus viscosity and mucociliary clearance. In general, a decrease in pH makes mucus more viscoelastic and slows its clearance. - We wanted to evaluate whether HCO3 secretion increases using porcine tracheal mucosa and pH stat. - When HCO3 production was evaluated, 10 μM formoterol + 0.3 μM methacholine -The secretion rate increased by 68% (µM / cm2 / hr) (baseline: 0.76±0.11, 1.24±0.14) (Figure 22). - Synergistic agonists increase HCO3 secretion compared to baseline conditions. - It significantly increased the secretion rate (p=2E-05, n=13 from 8 pig tracheas).

[0126] Synergistic agonists increased ASL height in ex vivo WT pig trachea. Synchrotron-based phase contrast imaging (PCI) was used to measure ASL height in porcine tracheal specimens. The synchrotron beamline generates X-rays with sufficient parallel spatial coherence to enhance the contrast between the airway lumen and the ASL layer by PCI. As X-rays pass through intact bronchi, the difference in refractive index between the ASL and airway lumen causes a phase shift in the X-rays, generating a characteristic interference pattern that appears as a change in X-ray intensity on a charge-coupled device (CCD) detector. ASL height measurements were obtained every 5 min after instillation of agarose beads, which served as a measurement barometer (T15 - baseline, T30 - 10 μM formoterol only, followed by T30 - 0.3 μM methacholine + 10 μM formoterol). As shown in Figure 23A, ASL height increased in both β-adrenergic and combined synergistic agonist-treated pigs (n = 4 pig tracheas), whereas vehicle-treated (DMSO-treated) control pigs (n = 2 pig tracheas) did not increase ASL height during the same measurement period. The mean rate of increase in ASL height during the synergistic agent-treated period was significantly higher than that during the formoterol-treated period (P < 0.005, n = 4 pigs) (μm / min). Baseline (T5-T15), 0.5, 10 μM formoterol (T30-T50), 1.7 ± 0.1; synergistic agonist (T65-T85), 3.6 ± 0.4 (Figure 23B).

[0127] Although the foregoing invention has been described in some detail by way of illustration and example for ease of understanding, it will be readily apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0128] Accordingly, the foregoing merely illustrates the principles of the present invention. It will be appreciated that those skilled in the art will be able to devise various configurations, not explicitly described or shown herein, which embody the principles of the present invention and are within the spirit and scope of the present invention. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts to which the inventors have contributed to the development of the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., any elements developed to perform the same function, regardless of structure. Furthermore, nothing disclosed herein, whether or not explicitly recited in a claim, is intended to be publicly disclosed.

[0129] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined to apply to a claim limitation only if the precise phrase "means for" or "step" is recited at the beginning of the claim limitation. If such precise phrase is not used in a claim limitation, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) does not apply.

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[0131] Notwithstanding any appended claims, the disclosure set forth herein is also described by the following clauses. 1. A method of treating an individual with a mucus obstructive disorder, comprising: Administering a β-adrenergic agonist or an adenylate cyclase activator in combination with a cholinergic agonist to an individual to treat a mucus obstructive disorder. 2. The method of paragraph 1, wherein the mucosal obstructive disease is selected from the group consisting of cystic fibrosis, primary ciliary motility disease, asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, and chronic bronchitis. 3. The method of claim 1, wherein the β-adrenergic agonist is a β2-adrenergic agonist. 4. The method of claim 3, wherein the β2-adrenergic agonist is selected from the group consisting of formoterol, albuterol, isoproterenol, pirbuterol, levalbuterol, clenbuterol, salmeterol, indacaterol, and vilanterol. 5. The method of claim 1, wherein the adenylate cyclase activator is forskolin or colforsin. 6. The method according to any one of items 1 to 5, wherein the cholinergic agonist is a direct-acting cholinergic agonist. 7. The method of claim 6, wherein the direct acting cholinergic agonist is selected from the group consisting of methacholine, acetylcholine, bethanechol, pilocarpine, and carbachol. 8. The method of any one of paragraphs 1 to 7, wherein the β-adrenergic agonist and the cholinergic agonist are administered sequentially. 9. The method of paragraph 8, wherein the β-adrenergic agonist is administered before the cholinergic agonist. 10. The method of any one of clauses 1 to 9, wherein the β-adrenergic agonist and the cholinergic agonist are administered simultaneously. 11. The method of any one of paragraphs 1 to 10, wherein the β-adrenergic agonist and the cholinergic agonist are administered systemically. 12. The method of any one of paragraphs 1 to 10, wherein the β-adrenergic agonist and the cholinergic agonist are administered locally. 13. The method of any one of paragraphs 1 to 12, wherein administration does not cause constriction of airway smooth muscle. 14. The method of any one of paragraphs 1 to 13, further comprising administering one or more cystic fibrosis transmembrane conductance regulator (CFTR) modulators. 15. The method of paragraph 14, wherein the one or more CFTR modulators are elexacaftar, tezacaftar, dutivacaftar, banzacaftar, and ivacaftar. 16. The method of any one of paragraphs 1-15, wherein administration results in a synergistic increase in mucus transport compared to either agonist used alone. 17. The method of any one of paragraphs 1 to 16, wherein the individual is a human. 18. A method for increasing airway submucosal gland secretion rate in an individual, comprising: Administering a beta-adrenergic agonist or an adenylate cyclase activator to an individual in combination with a cholinergic agonist to increase the rate of airway submucosal gland secretion in the individual. 19. The method of claim 18, wherein the β-adrenergic agonist is a β2-adrenergic agonist. 20. The method of claim 19, wherein the β2-adrenergic agonist is selected from the group consisting of formoterol, albuterol, isoproterenol, pirbuterol, levalbuterol, clenbuterol, salmeterol, indacaterol, and vilanterol. 21. The method of claim 18, wherein the adenylate cyclase activator is forskolin or colforsin. 22. The method of any one of clauses 18 to 21, wherein the cholinergic agonist is a direct acting cholinergic agonist. 23. The method of paragraph 22, wherein the direct-acting cholinergic agonist is selected from the group consisting of methacholine, acetylcholine, bethanechol, pilocarpine, and carbachol. 24. The method of any one of paragraphs 18 to 23, wherein the β-adrenergic agonist and the cholinergic agonist are administered sequentially. 25. The method of any one of clauses 18 to 23, wherein the β-adrenergic agonist and the cholinergic agonist are administered simultaneously. 26. The method of any one of paragraphs 18 to 25, wherein the β-adrenergic agonist and the cholinergic agonist are administered systemically. 27. The method of any one of paragraphs 18 to 26, wherein administration synergistically increases the submucosal gland secretion rate compared to the submucosal gland secretion rate when either agonist is used alone. 28. The method of any one of paragraphs 18 to 27, wherein the individual is a mammal. 29. The method of any one of paragraphs 18 to 27, wherein the individual is a pig. 30. The method of any one of paragraphs 18 to 27, wherein the individual is a ferret. 31. The method of any one of paragraphs 18 to 27, wherein the individual is a human. 32. The method of any one of clauses 18-31, wherein the individual has a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in non-functional, reduced function of the CTFR protein, or the absence of the CFTR protein. 33. A method for inhibiting cholinergic agonist-induced airway smooth muscle contraction in an individual, comprising: Administering a β-adrenergic agonist or an adenylate cyclase activator to an individual, wherein the administration of the β-adrenergic agonist or adenylate cyclase activator occurs before or simultaneously with the administration of a cholinergic agonist to the individual to inhibit contraction of airway smooth muscle. 34. The method of claim 33, wherein the β-adrenergic agonist is a β2-adrenergic agonist. 35. The method of claim 34, wherein the β2-adrenergic agonist is selected from the group consisting of formoterol, albuterol, isoproterenol, pirbuterol, levalbuterol, clenbuterol, salmeterol, indacaterol, and vilanterol. 36. The method of claim 35, wherein the adenylate cyclase activator is forskolin or colforsin. 37. The method of any one of clauses 34 to 36, wherein the cholinergic agonist is a direct acting cholinergic agonist. 38. The method of paragraph 37, wherein the direct-acting cholinergic agonist is selected from the group consisting of methacholine, acetylcholine, bethanechol, pilocarpine, and carbachol. 39. The method of any one of clauses 34 to 38, wherein the β-adrenergic agonist is administered systemically. 40. The method of any one of paragraphs 34 to 38, wherein the β-adrenergic agonist is administered locally. 41. The method of any one of paragraphs 34 to 40, wherein the individual is a mammal. 42. The method of any one of paragraphs 34 to 40, wherein the individual is a pig. 43. The method of any one of paragraphs 34 to 40, wherein the individual is a ferret. 44. The method of any one of paragraphs 34 to 40, wherein the individual is a human. 45. The method of any one of items 34 to 44, wherein the cystic fibrosis transmembrane conductance regulator (CFTR) gene has a mutation that results in the CTFR protein being non-functional or the functionality of the CTFR protein being reduced. 46. ​​A pharmaceutical composition comprising: β-adrenergic agonists or adenylate cyclase activators, Cholinergic agonists, and Pharmaceutical excipients. 47. The method of claim 46, wherein the β-adrenergic agonist is a β2-adrenergic agonist. 48. The method of claim 47, wherein the β2-adrenergic agonist is selected from the group consisting of formoterol, albuterol, isoproterenol, pirbuterol, levalbuterol, clenbuterol, salmeterol, indacaterol, and vilanterol. 49. The method of claim 46, wherein the adenylate cyclase activator is forskolin or colforsin. 50. The method of any one of clauses 46 to 49, wherein the cholinergic agonist is a direct acting cholinergic agonist. 51. The method of paragraph 50, wherein the direct-acting cholinergic agonist is selected from the group consisting of methacholine, acetylcholine, bethanechol, pilocarpine, and carbachol. 52. The method of any one of paragraphs 46 to 51, wherein the composition is formulated for systemic administration. 53. The method of any one of paragraphs 46 to 52, wherein the composition is formulated for topical administration. 54. The method of any one of paragraphs 46 to 53, wherein the composition is a solid composition. 55. The method of any one of paragraphs 46 to 53, wherein the composition is a liquid composition. 56. A device comprising the composition described in any one of paragraphs 46 to 55. 57. The device according to paragraph 56, wherein the device is an oral inhaler. 58. The device according to paragraph 56, wherein the device is a nasal inhaler.

[0132] In at least some of the foregoing embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment unless such substitution is technically feasible. Those skilled in the art will recognize that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to be included within the scope of the subject matter defined in the appended claims.

[0133] In general, those skilled in the art will understand that the terms used herein, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "comprising" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "comprising" should be interpreted as "including, but not limited to," etc.). Furthermore, those skilled in the art will understand that if a specific number of introduced claim recitations is intended, such intention will be explicitly set forth in the claim, and that the absence of such recitation indicates no such intention. For example, to aid in understanding, the following appended claims may use the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be interpreted as meaning that the introduction of a claim recitation by the indefinite article "a" or "an" limits a particular claim that includes the introduced claim recitation to embodiments that include only one such recitation. This is true even if the same claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). The same applies to the use of definite articles used to introduce claim recitations. Moreover, even when a specific number of introduced claims is explicitly recited, those of skill in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the mere recitation of "two recitations" without other modifiers means at least two recitations, or two or more recitations). Furthermore, when a rule similar to "at least one of A, B, C, etc." is used, such an interpretation is generally intended in the sense that one of skill in the art would understand the rule (e.g., "a system including at least one of A, B, C" includes, but is not limited to, systems including A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, C together, etc.).When a convention similar to "at least one of A, B, C, etc." is used, such configuration is generally intended in the sense that one of ordinary skill in the art would understand that convention (e.g., "a system including at least one of A, B, and C" includes, but is not limited to, systems including A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Furthermore, one of ordinary skill in the art will understand that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either term, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0134] Furthermore, where features or aspects of the disclosure are described in terms of a Markush group, one skilled in the art will recognize that the disclosure also is described in terms of any individual element or subgroup of elements of the Markush group.

[0135] As will be understood by those skilled in the art, for all purposes, including in terms of providing a written description, all ranges disclosed herein include all possible subranges and combinations of subranges. It is readily apparent that any listed range fully expresses and allows for the division of that same range into at least one half, third, quarter, fifth, tenth, etc. As a non-limiting example, each range described herein can be easily broken down into a lower third, middle third, upper third, etc. Those skilled in the art will also understand that all terms such as "up to," "at least," "greater than," "less than," etc., are inclusive of the recited numbers and refer to ranges that can be subsequently divided into subranges as described above. Finally, as will be understood by those skilled in the art, ranges include each individual element. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items.

[0136] Although the foregoing invention has been described in some detail by way of illustration and example for ease of understanding, it will be readily apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0137] Accordingly, the foregoing merely illustrates the principles of the present invention. It will be appreciated that those skilled in the art will be able to devise various configurations, not explicitly described or shown herein, which embody the principles of the present invention and are within the spirit and scope of the present invention. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts to which the inventors have contributed to the development of the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., any elements developed to perform the same function, regardless of structure. Furthermore, nothing disclosed herein, whether or not explicitly recited in a claim, is intended to be publicly disclosed.

[0138] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined to apply to a claim limitation only if the precise phrase "means for" or "step" is recited at the beginning of the claim limitation. If such precise phrase is not used in a claim limitation, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) does not apply.

Claims

1. 1. A method of treating an individual with a mucus obstructive disorder, comprising: Administering a β-adrenergic agonist or an adenylate cyclase activator in combination with a cholinergic agonist to an individual to treat a mucus obstructive disorder.

2. 2. The method of claim 1, wherein the mucosal obstructive disease is selected from the group consisting of cystic fibrosis, primary ciliary dyskinesia, asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, chronic bronchitis, and non-cystic fibrosis bronchiectasis.

3. β-adrenergic agonists are 2 - the method of claim 1, wherein the compound is an adrenergic agonist.

4. β 2 4. The method of claim 3, wherein the adrenergic agonist is selected from the group consisting of formoterol, albuterol, isoproterenol, pirbuterol, levalbuterol, clenbuterol, salmeterol, indacaterol, and vilanterol.

5. 2. The method of claim 1, wherein the adenylate cyclase activator is forskolin or colforsin.

6. The method according to any one of claims 1 to 5, wherein the cholinergic agonist is a direct acting cholinergic agonist.

7. 7. The method of claim 6, wherein the direct acting cholinergic agonist is selected from the group consisting of methacholine, acetylcholine, bethanechol, pilocarpine, and carbachol.

8. The method of any one of claims 1 to 7, wherein the β-adrenergic agonist and the cholinergic agonist are administered sequentially.

9. 9. The method of claim 8, wherein the β-adrenergic agonist is administered before the cholinergic agonist.

10. The method of any one of claims 1 to 9, wherein the β-adrenergic agonist and the cholinergic agonist are administered simultaneously.

11. The method of any one of claims 1 to 10, wherein the β-adrenergic agonist and the cholinergic agonist are administered systemically.

12. The method of any one of claims 1 to 10, wherein the β-adrenergic agonist and the cholinergic agonist are administered locally.

13. The method of any one of claims 1 to 12, wherein administration does not cause contraction of airway smooth muscle.

14. 14. The method of any one of claims 1 to 13, further comprising administering one or more cystic fibrosis transmembrane conductance regulator (CFTR) modulators.

15. 15. The method of claim 14, wherein the one or more CFTR modulators are elexacaftar, tezacaftar, and ivacaftar.

16. 16. The method of any one of claims 1 to 15, wherein administration results in a synergistic increase in mucus transport compared to either agonist used alone.

17. The method of any one of claims 1 to 16, wherein the individual is a human.

18. A pharmaceutical composition comprising: β-adrenergic agonists or adenylate cyclase activators, Cholinergic agonists, and Pharmaceutical excipients.

19. 19. The method of claim 18, wherein the beta-adrenergic agonist or adenylate cyclase activator is selected from the group consisting of formoterol, albuterol, isoproterenol, pirbuterol, levalbuterol, clenbuterol, salmeterol, indacaterol, vilanterol, forskolin, and colforsin.

20. 20. The method of claim 19, wherein the direct acting cholinergic agonist is selected from the group consisting of methacholine, acetylcholine, bethanechol, pilocarpine, and carbachol.

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